Cyclic polypeptides comprising a thioether linkage and methods for their preparation

ABSTRACT

This invention relates generally to cyclic polypeptides comprising a thioether linkage and methods for their preparation. More particularly, this invention relates to halogenated polypeptides having at least one haloalanine-like amino acid, and methods for their preparation which involve converting the hydroxyl group (i.e., --OH) of a serine-like amino acid to a halo group (i.e., --X where X is Cl, Br, or I) with the aid of a phosphorus-based halogenation reagent such as a triphenylphosphine dihalide (i.e., (C 6  H 5 ) 3  PX 2 , wherein X is Cl, Br, or I), a triphenylphosphite dihalide (i.e., (C 6  H 5  O) 3  PX 2 , wherein X is Cl, Br, or I), or a mixture of triphenylphosphine or triphenylphosphite with a halohydrocarbon (i.e., &#34;halo-conversion&#34;). This invention also relates to cyclic polypeptides having at least one polypeptide loop comprising a thioether linkage, and methods for their preparation which employ halogenated polypeptides and which involve intramolecular alkylation of the thiol group of a cysteine-like amino acid by the halo group of a haloalanine-like amino acid under suitable basic conditions to form a thioether linkage (i.e., &#34;cyclization&#34;).

This application is a continuation of application Ser. No. 08/660,739filed Jun. 6, 1996.

TECHNICAL FIELD

This invention relates generally to cyclic polypeptides comprising athioether linkage and methods for their preparation. More particularly,this invention relates to halogenated polypeptides having at least onehaloalanine-like amino acid, and methods for their preparation whichinvolve converting the hydroxyl group (i.e., --OH) of a serine-likeamino acid to a halo group (i.e., --X where X is Cl, Br, or I) with theaid of a phosphorus-based halogenation reagent such as atriphenylphosphine dihalide (i.e., (C₆ H₅)₃ PX₂, wherein X is Cl, Br, orI), a triphenylphosphite dihalide (i.e., (C₆ H₅)₃ PX₂, wherein X is Cl,Br, or I), or a mixture of triphenylphosphine or triphenylphosphite witha halohydrocarbon (i.e., "halo-conversion"). This invention also relatesto cyclic polypeptides having at least one polypeptide loop comprising athioether linkage, and methods for their preparation which employhalogenated polypeptides and which involve intramolecular alkylation ofthe thiol group of a cysteine-like amino acid by the halo group of ahaloalanine-like amino acid under suitable basic conditions to form athioether linkage (i.e., "cyclization").

DESCRIPTION OF THE RELATED ART

Throughout this application, various publications, patents, andpublished patent applications are referred to by an identifyingcitation. The disclosures of the publications, patents, and publishedpatent specifications referenced in this application are herebyincorporated by reference into the present disclosure to more fullydescribe the state of the art to which this invention pertains.

A thioether linkage has been widely utilized as a stable disulfidesurrogate to replace the native disulfide bridges of bioactive cyclicpeptides, such as hormones, neurotransmitters and neuromodulators, toprolong their biological activities (Lebl and Hruby, Tetrahedron Lett.(1984) 25:2067-2068; Polinsky et al., J. Med. Chem. (1992) 35:4185-4194;Mayer et al., Tetrahedron Lett. (1995) 36:7387-7390). The thioetherlinkage has also been used to prepare cyclic analogs of normally acyclicpolypeptides to restrict their conformational mobility and thus toincrease their biological activity and stability against biodegradation(Mosberg et al., J. Am. Chem. Soc. (1985) 107:2986-2987; Hruby et al.,Biochem. J. (1990) 268:249-262; Kataoka et al., Biopolymers (1992)32:1519-1533; Hruby and Bonner, Methods in Molecular Biology (1994)35:201-240).

Additionally, thioether linked cyclic peptides have also been found innature, especially in a family of polycyclic peptide antibiotics,lantibiotics, including nisin, an important food preservative,epidermin, a therapeutic agent against acne, as well as enzymeinhibitors and immunologically active peptides (Jung, G. Angew. Chem.Int. Ed. Engl. (1991) 30:1051-1192; Jack, R. W. and Sahl, H. G. Trend inBiotechnology (1995) 13:269-278; Sahl, H. G., Jack, R. W., and Bierbaum,G. Eur. J. Biochem. (1995) 230:827-853). Prominent structural featuresof all lantibiotics are intrachain sulfide bridges formed by thioetherdiaminodicarboxylic acids, lanthionines.

The conventional approach for the synthesis of thioether-linked cyclicpeptides utilizes thioether diamino acids lanthionines (e.g., H₂NCH(COOH)CH₂ SCH₂ CH(COOH)NH₂) and cystathionines (e.g., H₂ NCH(COOH)CH₂SCH₂ CH₂ CH(COOH)NH₂) as building blocks. The peptide cyclization isaccomplished through the formation of an amide bond (Lebl and Hruby,Tetrahedron Lett. (1984) 25:2067-2068; Osapay and Goodman, J. Chem. Soc.Chem. Commun. (1993):1599-1600; Safar et al., in Peptides: Chemistry,Structure and Biology (Hodges, R. S. and Smith, J. A., Eds.) Escom,Leiden, The Netherlands, (1994) 119-120). This approach requires tediousand extensive synthesis of orthogonally protected lanthionine andcystathionine derivatives (Jost and Rudinger, Collect. Czech. Chem.Commun. (1967) 32:2485-2490; Cavelier-Frontin et al. TetrahedronAsymmetry (1992) 3:85-94; Shao et al., J. Org. Chem. (1995)60:2956-2957; Probert et al., Tetrahedron Lett. (1996) 37:1101-1104).Recently, Rolinsky and co-workers have reported a synthetic approachwhich featured an intramolecular Michael addition of the thiol group ofa cysteine residue to an activated olefin to yield alanthionine-containing peptide (Polinsky et al., J. Med. Chem. (1992)35:4185-4194). However, this approach often yields two diastereomericproducts due to the lack of stereospecificity of Michael additionreaction (Probert et al., Tetrahedron Lett. (1996) 37:1101-1104). Mayerand co-workers have described a route which relies upon anintramolecular substitution reaction of bromo group by the thiol groupof cysteine residue to provide a cystathionine-containing peptide (Mayeret al., Tetrahedron Lett. (1995) 36:7387-7390). This approach is limitedby the low coupling efficiency of the bromo amino acid in the peptidesynthesis due to the competing intramolecular cyclization reaction. Thethioether bridge can also be formed through reversible sulfur extrusionwith tris(dialkyamino)phosphine (i.e., P(NR₂)₃) from the disulfidepeptides in moderate yields (Fukase et al., Bull. Chem. Soc. Jpn. (1985)59:2505-2508).

The present invention provides a general method for the halogenation ofpolypeptides. The present invention also provides a general method forthe use of halogenated polypeptides in the formation of cyclicpolypeptides comprising a thioether linkage. This synthetic methodcircumvents some of the limitations of earlier approaches and provides arobust method for the synthesis of thioether cyclic peptides.

This synthetic method may be used to build thioether constrained cyclicpeptide libraries to develop novel enzyme inhibitors, and agonists andantagonists of bioactive molecules (Katz et al., J. Am. Chem. Soc.(1995) 117:8541-8547). More particularly, the lanthionine-containinglibrary may be used to develop novel antimicrobial agents to combatantibiotic-resistant bacteria (Jung, Angew. Chem. Int. Ed. Engl. (1991)30:1051-1192; Blondelle and Houghten, Trends in Biotechnology (1996)14:60-65). The total synthesis of lantibiotics could also be greatlyfacilitated by the synthetic methods of the present invention.

The methods of the present invention may also be used to prepareconformationally restrained antigenic polypeptides. The cyclic thioetherantigens can be used to conjugate with immunogenic protein carriers orannular antigen scaffolds or to build multiple antigen peptides (MAP)(Dintzis, Pediatric Res. (1992) 32:356-376; Tam, Proc. Natl. Acad. Sci.USA. (1988) 85:5409-5413; Cunningham et al., United Kingdom patent GB 2282 813 (1995)). The peptide conjugates and multiple antigen peptides,which contain both a neutralizing B-cell epitope and a T-cell epitope,have been used as immunogens to effectively elicit vaccines againstvarious infectious diseases such as influenza, hepatitis B, and acquiredimmune deficiency syndrome (AIDS) (Tam, in Peptides: Synthesis,Structures, and Applications (Gutte ed.) Academic Press, San Diego,(1995) 455-500; Cunningham et al., United Kingdom patent GB 2 282 815(1995)).

In addition, the thioether cyclic antigens can be conjugated withmultivalent non-immunogenic platforms (Liu et al., Biochemistry (1979)18:690-697; Jones et al., Bioconjugate. Chem. (1994) 5:390-399; Jones etal., J. Med. Chem. (1995) 38:2138-2144). These peptide conjugatescontain only B-cell epitopes and could be used as toleragens fortreatment of antibody-mediated autoimmune diseases such as systematiclupus nephritis, anti-phospholipid antibody mediated thromboses,myasthenia gravis, Graves' disease and Rh hemolytic disease of newborns(Barstard and Iverson, U.S. Pat. No. 5,268,454 (1993); Coutts et al.,Lupus (1996)5:158-159).

One class of the cyclic polypeptides of the present invention,specifically, those with thioether-containing polypeptide loops of nineor fewer amino acids, or disulfide mimetics, bind to anticardiolipinantibody. These thioether cyclic polypeptides were derived from theirparent disulfide cyclic antiphospholipid epitopes whose primarysequences were obtained from phage display library screening (Victoriaand Marquis, U.S. patent application Ser. No. 08/482,651). Conjugates ofthese cyclic polypeptides may be used to suppress antiphospholipidantibodies to treat diseases such as recurrent stroke and recurrentfetal loss.

In addition to their applications in the synthesis of thioether cyclicpeptides, halopolypeptides are useful in the development of therapeuticagents such as enzyme inhibitors (Cheung et al., J. Med. Chem. (1983)26:1733-1741; Cheung et al., J. Med. Chem. (1986) 29:2060-2068) ordiagnostic reagents.

SUMMARY OF THE INVENTION

One aspect of the present invention pertains to cyclic polypeptideshaving at least one polypeptide loop, said loop comprising a thioetherlinkage, said cyclic polypeptide represented by the formula (SEQ IDNO:1): ##STR1## wherein S is a sulfur atom; L¹ and L² are independentlydivalent hydrocarbyl moieties of 1 to 10 carbon atoms; A¹ and A² areindependently alpha amino acid fragments; X¹ is represented by theformula J^(N) --(AA)_(p) --; X² is represented by the formula --(AA)_(q)--; X³ is represented by the formula --(AA)_(r) --J^(C) ; wherein AAdenotes an amino acid which may be in a protected form; J^(N) is anN-terminal group; J^(C) is a C-terminal group; and p, q, and r areindependently whole numbers from 0 to 50. In a preferred embodiment, thecyclic polypeptide is represented by the formula (SEQ ID NO:1): ##STR2##wherein S is a sulfur atom; C is a carbon atom; N is a nitrogen atom; Ois an oxygen atom; L¹ and L² are independently divalent hydrocarbylmoieties of 1 to 10 carbon atoms; R¹ and R² are independently --H or analkyl group having 1 to 6 carbon atoms; R¹ and R² are attached to carbonatoms, C, which independently have chirality R or S; R^(N1) and R^(N2)are independently --H or an alkyl group having 1 to 6 carbon atoms; X¹is represented by the formula J^(N) --(AA)_(p) --; X² is represented bythe formula --(AA)_(q) --; X³ is represented by the formula --(AA)_(r)--J^(C) wherein AA denotes an amino acid which may be in a protectedform; J^(N) is an N-terminal group; J^(C) is a C-terminal group; and p,q, and r are independently whole numbers from 0 to 50. In anotherpreferred embodiment, L¹ and L² are independently divalent alkylmoieties having from 1 to 6 carbon atoms, and more preferablyindependently selected from the group consisting of --CH₂ --, --CH₂ CH₂--, and --CH₂ CH₂ CH₂ --. In another preferred embodiment, p, q, and rare independently whole numbers from 0 to 10. In another preferredembodiment, R¹ and R² are independently --H or --CH₃. In anotherpreferred embodiment, R^(N1) and R^(N2) are independently --H or --CH₃.

Another aspect of the present invention pertains to halogenatedpolypeptides having at least one haloalanine-like amino acid, saidhalogenated polypeptide represented by the formula:

    Y.sup.1 --AA.sup.H --Y.sup.2

wherein AA^(H) is a haloalanine-like amino acid; Y¹ is represented bythe formula J^(N) --(AA)_(j) --; Y² is represented by the formula--(AA)_(k) --J^(C) wherein AA denotes an amino acid which may be in aprotected form; J^(N) is an N-terminal group; J^(C) is a C-terminalgroup; and j and k are independently whole numbers from 0 to 50, withthe proviso that j+k is not zero. In a preferred embodiment, thehalogenated polypeptide is represented by the formula:

    Y.sup.1 --NR.sup.N --CR.sup.H R.sup.B --C(═O)--Y.sup.2

wherein C is a carbon atom; N is a nitrogen atom; O is an oxygen atom;R^(H) is a halogen-containing alkyl group comprising a halo groupselected from the group consisting of --Cl, --Br, and --I; and an alkylmoiety of 1 to 10 carbon atoms; R^(B) is --H or an alkyl group having 1to 6 carbon atoms; R^(H) and R^(B) are attached to carbon atom, C, whichhas chirality R or S; R^(N) is --H or an alkyl group having 1 to 6carbon atoms; Y¹ is represented by the formula J^(N) --(AA)_(j) --; Y²is represented by the formula --(AA)_(k) --J^(C) ; wherein AA denotes anamino acid which may be in a protected form; J^(N) is an N-terminalgroup; J^(C) is a C-terminal group; and j and k are independently wholenumbers from 0 to 50, with the proviso that j+k is not zero. In anotherpreferred embodiment, R^(H) is a halogen-containing alkyl grouprepresented by the formula --(CH₂)_(z) X where z is a natural numberfrom 1 to 10 and X is Cl, Br, or I; more preferably selected from thegroup consisting of --CH₂ Cl, --CH₂ Br, --CH₂ CH₂ Cl, and --CH₂ CH₂ Br.In another preferred embodiment, j and k are independently whole numbersfrom 0 to 10. In another preferred embodiment, R^(B) is --H or --CH₃. Inanother preferred embodiment, R^(N) is --H or --CH₃.

Yet another aspect of the present invention pertains to methods for thepreparation of a cyclic polypeptide, said cyclic polypeptide having atleast one polypeptide loop, said loop comprising a thioether linkage;from a reactant polypeptide, said reactant polypeptide having at leastone cysteine-like amino acid, said cysteine-like amino acid having athiol group, and at least one serine-like amino acid, said serine-likeamino acid having an hydroxyl group; said method comprising the stepsof: (a) converting said hydroxyl group of said serine-like amino acid toa halo group with the aid of a phosphorus-based halogenation reagent toyield a haloalanine-like amino acid, and thus form a halogenatedpolypeptide; and (b) intramolecularly reacting said halo group of saidhaloalanine-like amino acid of said halogenated polypeptide with saidthiol group of said cysteine-like amino acid of said halogenatedpolypeptide under basic conditions to form said thioether linkage. In apreferred embodiment, said phosphorus-based halogenation reagentcomprises a reagent selected from the group consisting oftriphenylphosphine dihalide, triphenylphosphite dihalide, mixtures oftriphenylphosphine and a halohydrocarbon compound, and mixtures oftriphenylphosphite and a halohydrocarbon compound. In another preferredembodiment, said basic conditions are provided by the addition of sodiumcarbonate. In another preferred embodiment, said reactant polypeptide isprovided in a dissolved form. In another preferred embodiment, saidreactant polypeptide is provided in a supported form; said conversionstep (a) is performed using said supported reactant polypeptide; saidhalogenated polypeptide produced in step (a) is cleaved from its supportto yield a dissolved halogenated polypeptide, prior to carrying out step(b); and said reaction step (b) is performed using said dissolvedhalogenated polypeptide. In a preferred embodiment, said reactantpolypeptide is provided in a supported form; said conversion step (a) isperformed using said supported reactant polypeptide to yield a supportedhalogenated polypeptide; and said reaction step (b) is performed usingsaid supported halogenated polypeptide.

Still another aspect of the present invention pertains to methods forthe preparation of a halogenated polypeptide, said halogenatedpolypeptide having at least one haloalanine-like amino acid, saidhaloalanine-like amino acid having a halo group --X wherein X is Cl, Br,or I; from a reactant polypeptide, said reactant polypeptide having atleast one serine-like amino acid, said serine-like amino acid having anhydroxyl group; said method comprising the step: (a) converting saidhydroxyl group of said serine-like amino acid to a halo group with theaid of a phosphorus-based halogenation reagent to yield ahaloalanine-like amino acid. In a preferred embodiment, saidphosphorus-based halogenation reagent comprises a reagent selected fromthe group consisting of triphenylphosphine dihalide, triphenylphosphitedihalide, mixtures of triphenylphosphine and a halohydrocarbon compound,and mixtures of triphenylphosphite and a halohydrocarbon compound. Inanother preferred embodiment, a molar excess of said phosphorus-basedhalogenation reagent, in relation to said reactant polypeptide, isemployed. In another preferred embodiment, said hydroxyl group of saidserine-like amino acid is in a protected form; more preferably in aprotected form as a tert-butyldimethylsilyl ether group. In anotherpreferred embodiment, said reactant polypeptide is in a dissolved form.In another preferred embodiment, said reactant polypeptide is in asupported form.

As will become apparent, preferred features and characteristics of oneaspect of the invention are applicable to any other aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general synthetic strategy for a cyclic thioetherpolypeptide (SEQ ID NO:3).

FIG. 2 is a reaction scheme illustrating the synthesis of N^(a)-Fmoc-3G3-EMTE (SEQ ID NO:4) and 3G3-EMTE cyclic peptides as describedin Example 1.

FIG. 3 is a reaction scheme illustrating the synthesis of the 3G3-EMTE(SEQ ID NO:4) cyclic peptide as described in Example 2.

FIG. 4 is a reaction scheme illustrating the synthesis of the 3G3-MMTE(SEQ ID NO:3) cyclic peptides as described in Example 3.

FIG. 5 is a reaction scheme illustrating the synthesis of the 2G3-EMTE(SEQ ID NO:9) cyclic peptide as described in Example 5.

FIG. 6 is a reaction scheme illustrating the synthesis of the l-2G3-METEand d-2G3-METE (SEQ ID NO:10) cyclic peptides as described in Example 7.

FIG. 7 is a reaction scheme illustrating the synthesis of the l-2G3-METE(SEQ ID NO:10) cyclic peptide as described in Example 8.

FIG. 8 is a reaction scheme illustrating the synthesis of the G3-EETE(SEQ ID NO:16) cyclic peptide as described in Example 11.

FIG. 9 is a reaction scheme illustrating the synthesis of the AG3-EMTE(SEQ ID NO:19) cyclic peptide as described in Example 14.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Cyclic Polypeptides

The present invention pertains to cyclic polypeptides having at leastone polypeptide loop, wherein the polypeptide loop comprises a thioetherlinkage.

The term "polypeptide" is used herein in the conventional sense to referto a polymer of amino acids. The repeating units of a polypeptide arederived from amino acids and are chemically linked via an amide linkage(i.e., a peptide linkage; --C(═O)NR^(N) --, where R^(N) is a nitrogensubstituent, often --H). Polypeptides may be linear, branched, orcyclic, as determined by the chain of contiguous atoms (i.e., thepolypeptide backbone) which contains the peptide linkage atoms. The term"linear polypeptide" is used herein in the conventional sense to referto a polypeptide in which the polypeptide backbone is linear. The term"branched polypeptide" is used herein in the conventional sense to referto a polypeptide in which the polypeptide backbone comprises at leastone polypeptide branch. The term "cyclic polypeptide" is used herein inthe conventional sense to refer to a polypeptide in which thepolypeptide backbone comprises at least one polypeptide loop.

The term "thioether linkage" is used herein in the conventional sense torefer to a chemical linkage between two hydrocarbyl groups whichinvolves a single sulfur atom and is often denoted R--S--R.

Many of the cyclic polypeptides of the present invention mayconveniently be represented by the following formulae (SEQ ID NO:1):##STR3##

In the above formula (1), A¹ and A² denote amino acid fragments (oftendesignated herein as A^(F)) to which both the thioether linkage (i.e.,--L¹ --S--L² --) and the peptide fragment (i.e., --X² --) are attached,thus forming a polypeptide loop. The amino acid fragments, A¹ and A²,together with their associated linker moieties, L¹ and L², respectively,represent amino acid residues.

In the above formulae (I) and (II), L¹ and L² denote linker moieties andS denotes a sulfur atom joining the two linker moieties, thus forming athioether linkage (i.e., L¹ --S--L²). The linker moieties L¹ and L² areindependently divalent hydrocarbyl moieties. The term "hydrocarbylmoiety" is used herein in the conventional sense to refer to chemicalmoieties consisting of hydrogen (i.e., H) and carbon (i.e., C). Morepreferably, the linker moieties L¹ and L² are independently divalenthydrocarbyl moieties having from 1 to 10 carbon atoms; still morepreferably linear, cyclic, or branched divalent alkyl moieties havingfrom 1 to 10 carbon atoms. Preferred linker moieties L¹ and L² aredivalent alkyl moieties having from 1 to 6 carbon atoms, including, forexample, --CH₂ -- (i.e., methylene), --CH₂ CH₂ -- (i.e., ethylene), and--CH₂ CH₂ CH₂ -- (i.e., n-propylene). For convenience, the thioetherlinkage --CH₂ --S--CH₂ -- is denoted herein as MMTE (i.e.,methylene-methylene-thioether); the thioether linkage --CH₂ CH₂ --S--CH₂-- is denoted herein as EMTE (i.e., ethylene-methylene-thioether); thethioether linkage --CH₂ --S--CH₂ CH₂ -- is denoted herein as METE (i.e.,methylene-ethylene-thioether); and the thioether linkage --CH₂ CH₂--S--CH₂ CH₂ -- is denoted herein as EETE (i.e.,ethylene-ethylene-thioether).

In the above formula (II), C, N and O denote carbon, nitrogen, andoxygen atoms, respectively, and R¹ and R² denote substituents which areindependently --H or an organic substituent. In a preferred embodiment,R¹ and R² are independently --H or an alkyl group having 1 to 6 carbonatoms. In another preferred embodiment, R¹ and R² are independently --Hor --CH₃. In still another preferred embodiment, both R¹ and R² are --H.The chiralities at these two carbons (i.e., denoted C with R¹ and R²substituents, respectively) are independently R or S.

In the above formula (II), R^(N1) and R^(N2) denote nitrogensubstituents which may independently be --H or an organic substituent.Examples of organic substituents include those found in N^(a) -alkylalpha amino acids, such as alkyl groups having 1 to 6 carbon atoms,including for example, --CH₃. Other examples of organic substituentsinclude those found in cyclic alpha amino acids, such as, for example,proline (i.e., Pro), tetrahydroisoquinolinecarboxylic acid (i.e., Tic)and tetrahydrocarbolinecarboxylic acid (i.e., Tca), as described below.

In some embodiments, one or more of the substituents R¹, L¹, and R^(N1)may together form a single multivalent substituent. Similarly, one ormore of the substituents R², L², and R^(N2) may together form a singlemultivalent substituent. Thus, linker moieties may be multiply attachedto the polypeptide. For example, when the amino acid A¹ (or A²) isderived from an amino acid such as 4-mercaptoproline, the substituentsL¹ and R^(N1) together form a single trivalent substituent (i.e., --CH₂CH(--)CH₂ --) which links the alpha carbon atom, the amino nitrogenatom, and the thioether sulfur atom. In another example, when the aminoacid A¹ (or A²) is derived from an amino acid such as1-amino-3-mercapto-1-cyclopentane carboxylic acid (i.e., an analog ofcyclic leucine, Ac₅ c), the substituents L¹ and R¹ together form asingle trivalent substituent (e.g.,--CH₂ CH(--)CH₂ CH₂ --) which linksthe alpha carbon atom (twice) and the thioether sulfur atom.

In the above formula (I), X¹, X², X³ are peptide fragments which may berepresented by the formulae J^(N) --(AA)_(p) --, --(AA)_(q) --, and--(AA)_(r) --J^(C), respectively, wherein AA denotes an amino acid;J^(N) is an N-terminal group; J^(C) is a C-terminal group; and p, q andr are independently whole numbers, preferably from 0 to about 50, morepreferably from 0 to about 20, yet more preferably from 0 to about 10.The polypeptide fragments --(AA)_(p) --, --(AA)_(q) --, and --(AA)_(r)--, when present (i.e., when p, q, and/or r are non-zero), mayindependently be linear, branched, or cyclic, but preferably are linear.In a preferred embodiment, q is 7 or less and the polypeptide loopconsists of nine or fewer amino acid residues. In a preferredembodiment, the amino acids, AA, are alpha amino acids. The amino acids,AA, may be in a protected form or an unprotected form.

The N-terminal group, J^(N), identified above may be --H or a suitableterminal group. Examples of N-terminal groups, J^(N), include --H(yielding a free amino group); carboxy groups (i.e., --C(═O)OR, yieldinga carbamate group); and carbonyl groups (i.e., --C(═O)R; yielding anacyl amino group). Examples of carboxy groups include --Fmoc (i.e.,9-fluorenylmethyloxycarbonyl), --Boc (i.e., tert-butoxycarbonyl,--C(═O)OC(CH₃)₃), --CBZ (i.e., benzyloxycarbonyl, --C(═O)OCH₂ C₆ H₅),and --2-Cl--CBZ (i.e., 2-chlorobenzyloxycarbonyl, --C(═O)OCH₂ C₆ H₄ Cl).Examples of carbonyl groups include alkyl carbonyls of 1 to 10 carbonatoms, such as, acetyl (i.e., --C(═O)CH₃).

The C-terminal group, J^(C), identified above may be --H or a suitableterminal group. Examples of C-terminal groups, J^(N), include hydroxyl(i.e., --OH; yielding a free carboxylic acid group); alkoxy groups(i.e., --OR; yielding an ester group); amino groups (i.e., --NH₂, NHR,NR₂ ; yielding an amide group); and hydrazino groups (e.g., --NHNH₂ ;yielding a hydrazide group). Examples of alkoxy groups include alkoxygroups of 1 to 10 carbon atoms, such as methoxy (i.e., --OCH₃), ethoxy(i.e., --OCH₂ CH₃), cyclohexyloxy (i.e., --OcHx; --OC₆ H₁₁), tert-butoxy(i.e., --OC(CH₃)₃); and benzyloxy (i.e., --OCH₂ C₆ H₅). Examples ofamino groups include primary alkyl amino groups (i.e., --NHR; yielding asecondary amide group) and secondary alkyl amino groups (i.e., --NR₂ ;yielding a tertiary amide group) where R may independently be an alkylgroup of 1 to 10 carbon atoms, such as methyl (i.e., --CH₃) and ethyl(i.e., --CH₂ CH₃).

The term "amino acid" is used herein in the conventional sense to referto an organic chemical species comprising at least one amino group(i.e., --NH₂ or --NR^(N) H) and at least one carboxylic acid group(i.e., --COOH). In some cases, an amino group may be a substituted aminogroup (i.e., --NR^(N) H, where R^(N) is a nitrogen substituent), forexample, as in the case of proline. For convenience, amino acids areoften denoted herein as AA, or as H--AA--OH, where the initial --H ispart of an amino group, and the final --OH is part of a carboxylic acidgroup. Amino acids may often be conveniently further classifiedaccording to their structure, for example, as alpha-amino acids,beta-amino acids, and the like.

The term "alpha amino acid" is used herein the conventional sense torefer to amino acids in which at least one carboxylic acid group (i.e.,--COOH) and at least one amino group (i.e., --NH₂ or --NR^(N) H) aredirectly attached to a single carbon atom (designated the alpha carbon)and may be conveniently denoted HNR^(N) --CR^(A) R^(B) --COOH whereinR^(N), R^(A) and R^(B) are substituents. Two or more of the substituentsR^(N), R^(A) and R^(B) may together form a single multivalentsubstituent. For example, in the cyclic alpha-amino acid proline, R^(N)and R^(A) together form the single divalent substituent --CH₂ CH₂ CH₂--, and R^(B) is --H.

If the substituents R^(A) and R^(B) are different, the alpha carbon willbe chiral (i.e., R or S), and the alpha-amino acid will be opticallyactive. For example, glycine, for which R^(A) and R^(B) are both --H, isnot optically active, whereas alanine, for which R^(A) is --CH₃ andR^(B) is --H, is optically active and may be in d- or l-forms, denotedd-alanine or l-alanine, respectively. The alpha carbon of d-alanine isin the R configuration whereas the alpha carbon of l-alanine is in the Sconfiguration.

Of the wide variety of alpha-amino acids known, only about twenty arenaturally occurring. Naturally occurring alpha-amino acids are oftendenoted HNR^(N) --CHR--COOH (since R^(B) is --H) where R^(N) denotes anitrogen substituent and R denotes an amino acid substituent (oftenreferred to as an amino acid sidechain). The nitrogen substituent R^(N)is --H for all naturally occurring alpha amino acids, with the exceptionof proline (where R^(N) and R together form the divalent substituent--CH₂ CH₂ CH₂ --). Except for glycine, all of these twenty naturallyoccurring alpha-amino acids are optically active and are in the l-form.Examples of amino acid substituents include those substituents found inthe twenty naturally occurring alpha-amino acids, such as, for example,--H (for glycine), --CH₃ (for alanine), --CH₂ OH (for serine),--CH(CH₃)OH (for threonine), --CH₂ SH (for cysteine), and --CH₂ C₆ H₅(for phenylalanine). Other examples of amino acid substituents includethose which are structurally similar to those substituents found in thenaturally occurring amino acids, such as, for example, --CH₂ CH₂ OH (forhomoserine) and --CH₂ CH₂ SH (for homocysteine).

For convenience, the naturally occurring amino acids are oftenrepresented by a three-letter code or a one-letter code. For example,cysteine is often abbreviated as H--Cys--OH, or H--C--OH, and serine isoften abbreviated as H--Ser--OH or H--S--OH wherein the --H group ispart of the amino group (i.e., --NH₂ or --NR^(N) H) and the --OH groupis part of the carboxylic acid group (i.e., --COOH). Often the --H and--OH groups are omitted for the sake of simplicity, as in, for exampleCys, C; and Ser, S. Three-letter and one-letter codes for the twentynaturally occurring acids are well established in the art, and the sameconvention is used herein. As used herein, the corresponding "one-lettercode" for homoserine is Hs and the corresponding "one-letter code" forhomocysteine is Hc.

In addition to an alpha carboxylic acid group (i.e., --COOH) and analpha amino group (i.e., --NH₂ or --NR^(N) H), many amino acids haveadditional functional groups. Lysine, for which the amino acidsubstituent, R, is --(CH₂)₄ NH₂, has an additional amino group (i.e.,--NH₂). Aspartic acid and glutamic acid, for which the amino acidsubstituents, R, are --CH₂ COOH and --(CH₂)₂ COOH, respectively, eachhave an additional carboxylic acid group (i.e., --COOH). Serine, forwhich the amino acid substituent, R, is --CH₂ OH, has an additionalprimary hydroxyl group (i.e., --OH). Threonine, for which the amino acidsubstituent, R, is --CH(CH₃)OH, has an additional secondary hydroxylgroup (i.e., --OH). Cysteine, for which the amino acid substituent, R,is --CH₂ SH, has an additional thiol group (i.e., --SH). Other aminoacids have other additional functional groups, including, for example,thioether groups (e.g., in methionine), phenol groups (e.g., intyrosine), amide groups (e.g., in glutamine), and heterocylic groups(e.g., in histidine).

In addition to the twenty naturally occurring amino acids, several otherclasses of alpha amino acids are also known. Examples of these otherclasses include d-amino acids, N^(a) -alkyl amino acids, alpha-alkylamino acids, cyclic amino acids, chimeric amino acids, and miscellaneousamino acids. These non-natural amino acids have been widely used tomodify bioactive polypeptides to enhance resistance to proteolyticdegradation and/or to impart conformational constraints to improvebiological activity (Hruby et al., Biochem. J. (1990) 268:249-262; Hrubyand Bonner, Methods in Molecular Biology (1994) 35:201-240). The mostcommon N^(a) -alkyl amino acids are the N^(a) -methyl amino acids, suchas, N^(a) -methyl glycine (i.e., N^(a) MeGly), N^(a) -methyl alanine(i.e., N^(a) MeAla), and N^(a) -methyl lysine (i.e., N^(a) MeLys).Examples of alpha-alkyl amino acids include alpha-aminoisobutyric acid(i.e., Aib), diethylglycine (i.e., Deg), diphenylglycine (i.e., Dpg),alpha-methyl proline (i.e., (αMe)Pro), and alpha-methyl valine (i.e.,(αMe)Val) (Balaram, Pure & Appl. Chem. (1992) 64:1061-1066; Toniolo etal., Biopolymers (1993) 33:1061-1072; Hinds et al., J. Med. Chem. (1991)34:1777-1789). Examples of cyclic amino acids include1-amino-1-cyclopropane carboxylic acid (i.e., Ac₃ c),1-amino-1-cyclopentane carboxylic acid (i.e., cyclic leucine, Ac₅ c),aminoindane carboxylic acid (i.e., Ind),tetrahydroisoquinolinecarboxylic acid (i.e., Tic) andtetrahydrocarbolinecarboxylic acid (i.e., Tca) (Toniolo, C., Int. J.Peptide Protein Res. (1990) 35:287-300; Burgess, K., Ho, K. K., and Pal,B. J. Am. Chem. Soc. (1995) 117:3808-3819). Examples of chimeric aminoacids include penicillamine (i.e., Pen), combination of cysteine withvaline, and 4-mercaptoproline (i.e., Mpt), combination of proline andhomocysteine. Example of miscellaneous alpha-amino acids includeornithine (i.e., Orn), 2-naphthylalanine (i.e., 2-Nal), phenylglycine(i.e., Phg), t-butylglycine (i.e., tBug), cyclohexylalanine (i.e., Cha),and alpha-amino-2-thiophenepropionic acid (i.e., Thi). In addition toalpha-amino acids, others such as beta amino acids, can also be used inthe present invention. Examples of these other amino acids include2-aminobenzoic acid (i.e., Abz), β-aminopropanoic acid (i.e., β-Apr),γ-aminobutyric acid (i.e., γ-Abu), and 6-aminohexanoic acid (i.e.,ε-Ahx).

In the synthesis and manipulation of amino acid-containing species(e.g., polypeptides), it is often necessary to "protect" certainfunctional groups (such as alpha-amino groups, alpha-carboxylic acidgroups, and side-chain functional groups) of amino acids. A wide varietyof protecting groups and strategies are known in the art. For example,an alpha-amino group (i.e., --NH₂) may be protected with a9-fluorenylmethyloxycarbonyl group (i.e., Fmoc; as --NHFmoc), atert-butoxycarbonyl group (i.e., --C(═O)OC(CH₃)₃, Boc; as --NHBoc), or abenzyloxycarbonyl group (i.e., --C(═O)OCH₂ C₆ H₅, CBZ; as --NHCBZ). Theguanidino group of arginine (i.e., --NHC(═NH)NH₂) may be protected witha 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (i.e., Pmc; as--NHC(═NH)--NH--Pmc), a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group(i.e., Mtr; as --NHC(═NH)--NH--Mtr), or a mesitylene-2-sulfonyl group(i.e., Mts; as --NHC(═NH)--NH--Mts). The carboxamide groups ofasparagine and glutamine (i.e., --CONH₂) may be protected with a tritylgroup (i.e., --C(C₆ H₅)₃, Tr; as --CONHTr). The side chain carboxylicacid groups of aspartic and glutamic acid may be protected with at-butyl group (i.e., --C(CH₃)₃, tBu; as --COOtBu) or a cyclohexyl group(i.e., --C₆ H₁₁, cHx; as --COOcHx). Additionally, carboxylic acidgroups, such as terminal carboxylic acid groups, may be protected with amethyl group (i.e., --CH₃, as --COOCH₃), an ethyl group (i.e., --CH₂CH₃, as --COOCH₂ CH₃), or a benzyl group (i.e., --CH₂ C₆ H₅, as --COOCH₂C₆ H₅). The thiol group of cysteine (i.e., --SH) may be protected with at-butylthio group (i.e., --SC(CH₃)₃, tBuS; as --SStBu) or a trityl group(i.e., --C(C₆ H₅)₃, Tr; as --STr). The imidazole group of histidine maybe protected with a trityl group (i.e., --C(C₆ H₅)₃, Tr). Theepsilon-amino group of lysine (i.e., NH₂) may be protected with atert-butoxycarbonyl group (i.e., --C(═O)OC(CH₃)₃, Boc as --NHBoc), abenzyloxycarbonyl group (i.e., --C(═O)OCH₂ C₆ H₅, CBZ; as --NHCBZ), or a2-chlorobenzyloxycarbonyl group (i.e., --C(═O)OCH₂ C₆ H₄ Cl, 2-Cl--CBZ;as --NH--2-Cl--CBZ). The hydroxyl groups of homoserine, serine andthreonine (i.e., --OH) may be protected with a t-butyl group (i.e.,--C(CH₃)₃, tBu; as --OtBu), a trityl group (i.e., --C(C₆ H₅)₃, Tr; as--OTr), or a t-butyldimethylsilyl group (i.e., --Si(CH₃)₂ (C(CH₃)₃),TBDMS; as --OTBDMS). The indole nitrogen of tryptophan may be protectedwith a trityl group (i.e., --C(C₆ H₅)₃, Tr). The hydroxyl group oftyrosine (i.e., --OH) may be protected with a trityl group (i.e., --C(C₆H₅)₃, Tr; as --OTr).

The peptide linkage (i.e., --C(═O)--NRN--) of a polypeptide mayconveniently be considered to be the chemical linkage formed by reactinga carboxylic acid group (i.e., --COOH) of one amino acid with an aminogroup (i.e., --NR^(N) H) of another amino acid. In this way, apolypeptide (e.g., a "2-mer") of the two amino acids serine and cysteine(wherein the carboxylic acid group of serine and the amino group ofcysteine have formed a peptide linkage) may conveniently be representedas H--Ser--Cys--OH or H--S--C--OH, or, more simply, as Ser--Cys, S--C,or SC. The amino acid moieties of a polypeptide are often referred to asamino acid residues.

Examples of preferred cyclic polypeptides of the present inventioninclude those represented by formula (II) above which are thioetheranalogs of the disulfide polypeptide AGPCLGVLGKLCPG (denoted 3G3 (SEQ IDNO:2)) and wherein:

X¹ is Ala-Gly-Pro- (i.e., AGP-- and p is 3); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ --; L² is --CH₂ --; R² is--H; and R² is --H (denoted herein as compound 3G3-MMTE (SEQ ID NO:3)).The chirality of the carbon with substituent R¹ is mixed in d- andl-forms. The chirality of the carbon with the substituent R² is in thel-form.

X¹ is Ala-Gly-Pro- (i.e., AGP-- and p is 3); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ CH₂ --; L² is --CH₂ --; R¹is --H; and R² is --H (denoted herein as compound 3G3-EMTE (SEQ IDNO:4)). The chiralities of the carbons with substituents R¹ and R² arein the l-form.

X¹ is Ala-Gly-Pro- (i.e., AGP-- and p is 3); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ --; L² is --CH₂ CH₂ --; R¹is --H; and R² is --H (denoted herein as compound 3G3-METE (SEQ IDNO:5)). The chiralities of the carbons with substituents R¹ and R² arein the l-form.

X¹ is Ala-Gly-Pro- (i.e., AGP-- and p is 3); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ CH₂ --; L² is --CH₂ CH₂--; R¹ is --H; and R² is --H (denoted herein as compound 3G3-EETE (SEQID NO:6)). The chiralities of the carbons with substituents R¹ and R²are in the l-form.

Examples of preferred cyclic polypeptides of the present inventioninclude those represented by formula (II) above which are thioetheranalogs of the disulfide polypeptide GPCLGVLGKLCPG (denoted 2G3 (SEQ IDNO:7)) and wherein:

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ --; L² is --CH₂ --; R¹ is--H; and R² is --H (denoted herein as compound 2G3-MMTE (SEQ ID NO:8)).The chirality of the carbon with substituent R¹ is mixed in d- andl-forms. The chirality of the carbon with the substituent R² is in thel-form.

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ CH₂ --; L is --CH₂ --; R¹is --H; and R² is --H (denoted herein as compound 2G3-EMTE (SEQ IDNO:9)). The chiralities of the carbons with substituents R¹ and R² arein the l-form.

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ --; L is --CH₂ CH₂ --; R¹is --H; and R² is --H (denoted herein as compound 2G3-METE (SEQ IDNO:10)). The chirality of the carbon with substituent R¹ is in the d- orl-form. The chirality of the carbon with the substituent R² is in thel-form.

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., --LGVLGKL-- and q is 7); X³ is-Pro-Gly (i.e., --PG and r is 2); L¹ is --CH₂ CH₂ --; L² is --CH₂ CH₂--; R¹ is --H; and R² is --H (denoted herein as compound 2G3-EETE (SEQID NO:11)). The chiralities of the carbons with substituents R¹ and R²are in the l-form.

Examples of preferred cyclic polypeptides of the present inventioninclude those represented by formula (II) above which are thioetheranalogs of the disulfide polypeptide CLGVLGKLC (denoted G3 (SEQ IDNO:12)) and wherein:

X¹ is H-- (i.e., p is 0); X² is --Leu-Gly-Val-Leu-Gly-Lys-Leu-- (i.e.,--LGVLGKL-- and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ ; L² is--CH₂ --; R¹ is --H; and R² is --H (denoted herein as compound G3-MMTE(SEQ ID NO:13)). The chirality of the carbon with substituent R¹ ismixed in d- and l-forms. The chirality of the carbon with thesubstituent R² is in the l-form.

X¹ is H-- (i.e., p is 0); X² is --Leu-Gly-Val-Leu-Gly-Lys-Leu-- (i.e.,--LGVLGKL-- and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ CH₂ --;L² is --CH₂ --; R¹ is --H; and R² is --H (denoted herein as compoundG3-EMTE (SEQ ID NO:14)). The chiralities of the carbons withsubstituents R¹ and R² are in the l-form.

X¹ is H-- (i.e., p is 0); X² is --Leu-Gly-Val-Leu-Gly-Lys-Leu-- (i.e.,--LGVLGKL-- and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ --; L²is --CH₂ CH₂ --; R¹ is --H; and R² is --H (denoted herein as compoundG3-METE (SEQ ID NO:15)). The chiralities of the carbons withsubstituents R¹ and R² are in the l-form.

X¹ is H-- (i.e., p is 0); X² is --Leu-Gly-Val-Leu-Gly-Lys-Leu-- (i.e.,--LGVLGKL-- and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ CH₂ --;L² is --CH₂ CH₂ --; R¹ is --H; and R² is --H (denoted herein as compoundG3-EETE (SEQ ID NO:16)). The chiralities of the carbons withsubstituents R¹ and R² are in the l-form.

Examples of preferred cyclic polypeptides of the present inventioninclude those represented by formula (II) above which are thioetheranalogs of the disulfide polypeptide CLGVLAKLC (denoted AG3 (SEQ IDNO:17)) and wherein:

X¹ is H-- (i.e., p is 0); X² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-- (i.e., --L(N^(a) Me--G)(d-V)(d-L)AKL--and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ --; L² is --CH₂ --;R¹ is --H; and R² is --H (denoted herein as compound AG3-MMTE (SEQ IDNO:18)). The chirality of the carbon with substituent R¹ is mixed in d-and l-forms. The chirality of the carbon with the substituent R² is inthe l-form.

X¹ is H-- (i.e., p is 0); X² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-- (i.e., --L(N^(a) Me--G)(d-V)(d-L)AKL--and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ CH₂ --; L² is --CH₂--; R¹ is --H; and R² is --H (denoted herein as compound AG3-EMTE (SEQID NO:19)). The chiralities of the carbons with substituents R¹ and R²are in the l-form.

X¹ is H-- (i.e., p is 0); X² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-- (i.e., --L(N^(a) Me--G)(d-V)(d-L)AKL--and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ --; L² is --CH₂ CH₂--; R¹ is --H; and R² is --H (denoted herein as compound AG3-METE (SEQID NO:20)). The chiralities of the carbons with substituents R¹ and R²are in the l-form.

X¹ is H-- (i.e., p is 0); X² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-- (i.e., --L(N^(a) Me--G)(d-V)(d-L)AKL--and q is 7); X³ is --NH₂ (i.e., r is 0); L¹ is --CH₂ CH₂ --; L² is --CH₂CH₂ --; R¹ is --H; and R² is --H (denoted herein as compound AG3-EETE(SEQ ID NO:21)). The chiralities of the carbons with substituents R¹ andR² are in the l-form.

Examples of preferred cyclic polypeptides of the present inventioninclude those represented by formula (II) above which are thioetheranalogs of the disulfide polypeptide GPCLILAPDRC (denoted CB10 (SEQ IDNO:22)) and wherein:

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is--Leu-Ile-Leu-Ala-Pro-Asp-Arg-- (i.e., --LILAPDR-- and q is 7); X³ is--NH₂ (i.e., r is 0); L¹ is --CH₂ --; L² is --CH₂ --; R¹ is --H; and R²is --H (denoted herein as compound CB10-MMTE (SEQ ID NO:23)). Thechirality of the carbon with substituent R¹ is mixed in d- and l-forms.The chirality of the carbon with the substituent R² is in the l-form.

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is--Leu-Ile-Leu-Ala-Pro-Asp-Arg-- (i.e., --LILAPDR-- and q is 7); X³ is--NH₂ (i.e., r is 0); L¹ is --CH₂ CH₂ --; L² is --CH₂ --; R¹ is --H; andR² is --H (denoted herein as compound CB10-EMTE (SEQ ID NO:24). Thechiralities of the carbons with substituents R¹ and R² are in thel-form.

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is--Leu-Ile-Leu-Ala-Pro-Asp-Arg-- (i.e., --LILAPDR-- and q is 7); X³ is--NH₂ (i.e., r is 0); L¹ is --CH₂ --; L² is --CH₂ CH₂ --; R¹ is --H; andR² is --H (denoted herein as compound CB10-METE (SEQ ID NO:25)). Thechiralities of the carbons with substituents R¹ and R² are in thel-form.

X¹ is Gly-Pro- (i.e., GP-- and p is 2); X² is--Leu-Ile-Leu-Ala-Pro-Asp-Arg-- (i.e., --LILAPDR-- and q is 7); X³ is--NH₂ (i.e., r is 0); L¹ is --CH₂ CH₂ --; L² is --CH₂ CH₂ --; R¹ is --H;and R² is --H (denoted herein as compound CB10-EETE (SEQ ID NO:26)). Thechiralities of the carbons with substituents R¹ and R² are in thel-form.

B. Halogenated Polypeptides

The present invention also pertains to halogenated polypeptides havingat least one haloalanine-like amino acid, said haloalanine-like aminoacid having a halo group. The halogenated polypeptides may be in freeform (e.g., as a solid or in solution) or may be in a supported form(e.g., attached to a support material).

The term "haloalanine-like amino acid" is used herein to refer to alphaamino acids which may be represented by the formula HNR^(N) --CR^(H)R^(B) --COOH (as the free amino acid) or as --NR^(N) --CR^(H) R^(B)--C(═O)-- (when part of a polypeptide chain), where R^(N), R^(H) andR^(B) are substituents. The substituents R^(N) and R^(B) are as definedabove for R^(N1) /R^(N2) and R¹ /R², respectively, and are independently--H or an organic substituent. Two or more of the substituents R^(N),R^(H) and R^(B) may together form a single multivalent substituent. Thesubstituent R^(H) (or a single multivalent substituent incorporatingR^(H) and one or more of R^(N) and R^(B)) is a halogen-containing group.The term "halogen-containing group" is used herein to refer to organicmoieties which comprise a halo group (i.e., --X wherein X is Cl, Br, orI). The alpha carbon of the haloalanine-like amino acid may havechirality R or S.

In some preferred embodiments, R^(H) is a halogen-containing alkylgroup. The term "halogen-containing alkyl group" is used herein to referto organic moieties which comprise a halo group (i.e., --X wherein X isCl, Br, or I) and an alkyl moiety. Examples of preferred halo groups arethe bromo group (i.e., --Br) and the chloro group (i.e., --Cl). Thealkyl moiety preferably comprises from 1 to 10 carbon atoms, morepreferably 1 to 5 carbon atoms, still more preferably 1 to 3 carbonatoms, most preferably 1 to 2 carbon atoms. The alkyl moiety may belinear, cyclic, or branched, but is preferably linear. Examples ofpreferred halo-containing alkyl groups include those of the generalformula --(CH₂)_(z) X where z is a natural number from 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, most preferably 1 to 2,and X is Cl, Br, or I. Examples of preferred halo-containing alkylgroups include --CH₂ Cl, --CH₂ Br, --CH₂ CH₂ Cl, and --CH₂ CH₂ Br.Examples of other preferred halo-containing alkyl groups include--CH(CH₃)Cl and --CH(CH₃)Br.

Many of the halogenated polypeptides of the present invention mayconveniently be represented by the following formulae:

    Y.sup.1 --AA.sup.H --Y.sup.2                               (III)

    Y.sup.1 --NR.sup.N --CR.sup.H R.sup.B --C(═O)--Y.sup.2 (IV)

In the above formulae (III) and (IV), C, N, and O denote carbon,nitrogen, and oxygen atoms, respectively; AA^(H) denotes ahaloalanine-like amino acid as described above; and Y¹ and Y² denotepeptide fragments. Y¹ and Y² may be conveniently represented by theformulae J^(N) --(AA)_(j) -- and --(AA)_(k) --J^(C), respectively,wherein AA denotes an amino acid; J^(N) is an N-terminal group asdefined above; J^(C) is a C-terminal group as defined above; and j and kare independently whole numbers, preferably from 0 to about 50, morepreferably from 0 to about 20, yet more preferably from 0 to about 10;with the proviso that j+k is not zero. The peptide fragments --(AA)_(j)-- and --(AA)_(k) --, when present (i.e., when j and/or k are non-zero),may independently be linear, branched, or cyclic, but preferably arelinear. In some preferred embodiments, the amino acids, AA, are alphaamino acids. The amino acids, AA, may be in a protected form or anunprotected form.

Examples of preferred halogenated polypeptides of the present inventioninclude those represented by formula (IV) above which effectivelycomprise haloanalogs of the polypeptide AGPSLGVLGKLCPG (denoted X-3G3(SEQ ID NO:27)) and wherein:

Y¹ is Ala-Gly-Pro- (i.e., AGP-- and j is 3); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys-Pro-Gly-- (i.e., --LGVLGKLCPG-- and kis 10); R^(H) is --CH₂ X, wherein X is Cl, Br, or I; and R^(B) is --H(SEQ ID NO:28). The chirality of the carbon with substituents R^(H) andR^(B) is in the l-form.

Y¹ is Ala-Gly-Pro- (i.e., AGP-- and j is 3); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys-Pro-Gly-- (i.e., --LGVLGKLCPG-- and kis 10); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br, or I; and R^(B) is--H (SEQ ID NO:29). The chirality of the carbon with substituents R^(H)and R^(B) is in the l-form.

Y¹ is Ala-Gly-Pro- (i.e., AGP-- and j is 3); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine-Pro-Gly-- (i.e.,--LGVLGKLHcPG-- and k is 10); R^(H) is --CH₂ X, wherein X is Cl, Br, orI; and R^(B) is --H (SEQ ID NO:30). The chirality of the carbon withsubstituents R^(H) and R^(b) is in the l-form.

Y¹ is Ala-Gly-Pro- (i.e., AGP-- and j is 3); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine-Pro-Gly-- (i.e.,--LGVLGKLHcPG-- and k is 10); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br,or I; and R^(B) is --H (SEQ ID NO:31). The chirality of the carbon withsubstituents R^(H) and R^(B) is in the l-form.

Examples of preferred halogenated polypeptides of the present inventioninclude those represented by formula (IV) above which effectivelycomprise haloanalogs of the polypeptide GPSLGVLGKLCPG (denoted X-2G3(SEQ ID NO:32)) and wherein:

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys-Pro-Gly-- (i.e., --LGVLGKLCPG-- and kis 10); R^(H) is --CH₂ X, wherein X is Cl, Br, or I; and R^(B) is --H(SEQ ID NO:33). The chirality of the carbon with substituents R^(H) andR^(B) is in the l-form.

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys-Pro-Gly-- (i.e., --LGVLGKLCPG-- and kis 10); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br, or I; and R^(B) is--H (SEQ ID NO:34). The chirality of the carbon with substituents R^(H)and R^(B) is in the l-form.

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine-Pro-Gly-- (i.e.,--LGVLGKLHcPG-- and k is 10); R^(H) is --CH₂ X, wherein X is Cl, Br, orI; and R^(B) is --H (SEQ ID NO:35). The chirality of the carbon withsubstituents R^(H) and R^(B) is in the l-form.

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine-Pro-Gly-- (i.e.,--LGVLGKLHcPG-- and k is 10); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br,or I; and R^(B) is --H (SEQ ID NO:36). The chirality of the carbon withsubstituents R^(H) and R^(B) is in the l-form.

Y¹ is Gly-Pro-Cys-Leu-Gly-Val-Leu-Gly-Lys-Leu- (i.e., GPCLGVLGKL-- and jis 10); Y² is -Pro-Gly (i.e., --PG and k is 2); R^(H) is --CH₂ CH₂ X,wherein X is Cl, Br, or I; and R^(B) is --H (SEQ ID NO:37). Thechirality of the carbon with substituents R^(H) and R^(B) is in thel-form.

Examples of preferred halogenated polypeptides of the present inventioninclude those represented by formula (IV) above which effectivelycomprise haloanalogs of the polypeptide SLGVLGKLC (denoted X-G3 (SEQ IDNO:38)) and wherein:

Y¹ is H-- (i.e., j is 0); Y² is --Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys--NH₂(i.e., --LGVLGKLC--NH₂ and k is 8); R^(H) is --CH₂ X, wherein X is Cl,Br, or I; and R^(B) is --H (SEQ ID NO:40). The chirality of the carbonwith substituents R^(H) and R^(B) is in the l-form.

Y¹ is H-- (i.e., j is 0); Y² is --Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys--NH₂(i.e., --LGVLGKLC--NH₂ and k is 8); R^(H) is --CH₂ CH₂ X, wherein X isCl, Br, or I; and R^(B) is --H (SEQ ID NO:40). The chirality of thecarbon with substituents R^(H) and R^(B) is in the l-form.

Y¹ is H-- (i.e., j is 0); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine--NH₂ (i.e., --LGVLGKLHc--NH₂and k is 8); R^(H) is --CH₂ X, wherein X is Cl, Br, or I; and R^(B) is--H (SEQ ID NO:41). The chirality of the carbon with substituents R^(H)and R^(B) is in the l-form.

Y¹ is H-- (i.e., j is 0); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine--NH₂ (i.e., --LGVLGKLHc--NH₂and k is 8); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br, or I; and R^(B)is --H (SEQ ID NO:42). The chirality of the carbon with substituentsR^(H) and R^(B) is in the l-form.

Examples of preferred halogenated polypeptides of the present inventioninclude those represented by formula (IV) above which effectivelycomprise haloanalogs of the polypeptide SLGVLAKLC (denoted X-AG3 (SEQ IDNO:43)) and wherein:

Y¹ is H-- (i.e., j is 0); Y² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-Cys--NH₂ (i.e., --L(N^(a)Me--G)(d-V)(d-L)AKLC and k is 8); R^(H) is --CH₂ X, wherein X is Cl, Br,or I; and R^(B) is --H (SEQ ID NO:44). The chirality of the carbon withsubstituents R^(H) and R^(B) is in the l-form.

Y¹ is H-- (i.e., j is 0); Y² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-Cys--NH₂ (i.e., --L(N^(a)Me--G)(d-V)(d-L)AKLC and k is 8); R^(H) is --CH₂ CH₂ X, wherein X is Cl,Br, or I; and R^(B) is --H (SEQ ID NO:45). The chirality of the carbonwith substituents R^(H) and R^(B) is in the l-form.

Y¹ is H-- (i.e., j is 0); Y² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-homocysteine--NH₂ (i.e., --L(N^(a)Me--G)(d-V)(d-L)AKLHc and k is 8); R^(H) is --CH₂ X, wherein X is Cl,Br, or I; and R^(B) is --H (SEQ ID NO:46). The chirality of the carbonwith substituents R^(H) and R^(B) is in the l-form.

Y¹ is H-- (i.e., j is 0); Y² is --Leu-N^(a)MeGly-d-Val-d-Leu-Ala-Lys-Leu-homocysteine--NH₂ (i.e., --L(N^(a)Me--G)(d-V)(d-L)AKLHc and k is 8); R^(H) is --CH₂ CH₂ X, wherein X isCl, Br, or I; and R^(B) is --H (SEQ ID NO:47). The chirality of thecarbon with substituents R^(H) and R^(B) is in the l-form.

Examples of preferred halogenated polypeptides of the present inventioninclude those represented by formula (IV) above which effectivelycomprise haloanalogs of the polypeptide GPSLILAPDRC (denoted X-CB10) andwherein:

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Ile-Leu-Ala-Pro-Asp-Arg-Cys--NH₂ (i.e., --LILAPDRC--NH₂ and k is8); R^(H) is --CH₂ X, wherein X is Cl, Br, or I; and R^(B) is --H (SEQID NO:49). The chirality of the carbon with substituents R^(H) and R^(B)is in the l-form.

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-Cys--NH₂ (i.e., --LILAPDRC--NH₂ and k is8); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br, or I; and R^(B) is --H(SEQ ID NO:50). The chirality of the carbon with substituents R^(H) andR^(B) is in the l-form.

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine--NH₂ (i.e., --LILAPDRHc--NH₂and k is 8); R^(H) is --CH₂ X, wherein X is Cl, Br, or I; and R^(B) is--H (SEQ ID NO:51). The chirality of the carbon with substituents R^(H)and R^(B) is in the l-form.

Y¹ is Gly-Pro- (i.e., GP-- and j is 2); Y² is--Leu-Gly-Val-Leu-Gly-Lys-Leu-homocysteine--NH₂ (i.e., --LILAPDRHc--NH₂and k is 8); R^(H) is --CH₂ CH₂ X, wherein X is Cl, Br, or I; and R^(B)is --H (SEQ ID NO:52). The chirality of the carbon with substituentsR^(H) and R^(B) is in the l-form.

C. Preparation of Halogenated Polypeptides

The present invention also pertains methods for the preparation ofhalogenated polypeptides having at least one haloalanine-like aminoacid, said haloalanine-like amino acid having a halo group (i.e., --Xwherein X is Cl, Br, or I). More particularly, such halogenatedpolypeptides may be prepared from reactant polypeptides, said reactantpolypeptides having at least one serine-like amino acid, saidserine-like amino acid having an hydroxyl group (i.e., --OH). Morespecifically, the halogenated polypeptides of the present invention maybe prepared by converting the hydroxyl group of a serine-like amino acidto a halo group with the aid of a phosphorus-based halogenation reagent,thus yielding a haloalanine-like amino acid (i.e., "halo-conversion").

The term "serine-like amino acid" is used herein to refer to alpha aminoacids which may be represented by the formula HNR^(N) --CR^(O) R^(B)--COOH (as the free amino acid) or as --NR^(N) --CR^(O) R^(B) --C(═O)--(when part of a polypeptide chain), where R^(N), R^(O) and R^(B) aresubstituents. The substituents R^(N) and R^(B) are as defined above forR^(N1) /R^(N2) and R¹ /R², respectively, and are independently --H or anorganic substituent. Two or more of the substituents R^(N), R^(O) andR^(B) may together form a single multivalent substituent. Thesubstituent R^(O) (or a single multivalent substituent incorporatingR^(O) and one or more of R^(N) and R^(B)) is a hydroxyl-containinggroup. The term "hydroxyl-containing group" is used herein to refer toorganic moieties which comprise an hydroxyl group (i.e., --OH). Thealpha carbon of the serine-like amino acid may have chirality R or S.

In some preferred embodiments, R^(O) is an hydroxyl-containing alkylgroup. The term "hydroxyl-containing alkyl group" is used herein torefer to organic moieties which comprise an hydroxyl group (i.e., --OH)and an alkyl moiety. The alkyl moiety preferably comprises from 1 to 10carbon atoms, more preferably 1 to 5 carbon atoms, still more preferably1 to 3 carbon atoms, most preferably 1 to 2 carbon atoms. The alkylmoiety may be linear, cyclic, or branched, but is preferably linear.Examples of preferred hydroxyl-containing alkyl groups include those ofthe general formula --(CH₂)_(z) OH where z is a natural number from 1 to10, more preferably 1 to 5, still more preferably 1 to 3, mostpreferably 1 to 2. Examples of more preferred hydroxyl-containing alkylgroups include --CH₂ OH (i.e., as in the case of serine) and --CH₂ CH₂OH (i.e., as in the case of homoserine). Another example of a preferredhydroxyl-containing alkyl group is --CH(CH₃)OH (i.e., as in the case ofthreonine).

The hydroxyl group of the serine-like amino acid, which is to beconverted to a halo group, may be in a suitably protected form, or in afree form (i.e., as --OH). Preferably, the hydroxyl group is in aprotected form, as this may eliminate a deprotection step which mayotherwise be necessary, for example, when the reactant polypeptide isobtained in a protected form. Thus, in preferred embodiments, thehydroxyl group of the serine-like amino acid is protected, morepreferably with a TBDMS group (e.g., --Si(CH₃)₂ (C(CH₃)₃ ; as --OTBDMS).If it is desired to perform halo-conversion with the hydroxyl group ofthe serine-like amino acid in a free form (i.e., as --OH), the TBDMSgroup can be selectively removed with 3 equivalents of TBAF (i.e.,tetrabutylammonium fluoride) in THF (i.e., tetrahydrofuran) in thepresence of protecting groups other than base labile groups such asFmoc. Similarly, a trityl-protected hydroxyl group (i.e., --OTr) may beconveniently deprotected to yield the free hydroxyl group (i.e., --OH)with 1% TFA (i.e., trifluoroacetic acid) in 1:1 DCM/MeOH (i.e.,dichloromethane, methanol).

Halo-conversion is effected by reaction of the reactant polypeptide witha phosphorus-based halogenation reagent. As used herein, the term"phosphorus-based halogenation reagent" relates totrialkylphosphine-based or trialkylphosphite-based halogenationreagents. Examples of preferred halogenation reagents include thosecomprising triphenylphosphine dihalide (i.e., (C₆ H₅)₃ PX₂, wherein X isCl, Br, or I; dihalotriphenylphosphorane); triphenylphosphite dihalide(i.e., (C₆ H₅ O)₃ PX₂, wherein X is Cl, Br, or I); or a mixture oftriphenylphosphine (i.e., (C₆ H₅)₃ P) or triphenylphosphite (i.e., (C₆H₅ O)₃ P) with halohydrocarbon compounds. Examples of halohydrocarboncompounds include carbon tetrahalide (i.e., CX₄, wherein X is Cl, Br, orI), hexahaloacetone (i.e., CX₃ C(═O)CX₃, wherein X is independently Cl,Br, or I), and hexahaloethane (i.e., C₂ X₆, wherein X is independentlyCl, Br, or I). A preferred halogenation reagent comprisestriphenylphosphine dichloride (i.e., (C₆ H₅)₃ PCl₂). Another preferredhalogenation reagent comprises triphenylphosphine dibromide (i.e., (C₆H₅)₃ PBr₂). Yet another preferred halogenation reagent comprises amixture of triphenylphosphine (i.e., (C₆ H₅)₃ P) and carbontetrachloride (i.e., CCl₄).

Halo-conversion may be performed using a dissolved reactant polypeptide(i.e., in solution) or using a supported reactant polypeptide (e.g.,attached to a support material). For example, standard solid-phasepolypeptide synthesis methods may be used to obtain a desiredpolypeptide which is attached to a solid support. Halo-conversion maythen be performed using the supported polypeptide as the reactantpolypeptide, or alternatively, the polypeptide may be cleaved from thesupport and the conversion reaction may then be performed using thedissolved polypeptide as the reactant polypeptide.

In a preferred embodiment, halo-conversion is performed using asupported polypeptide as the reactant polypeptide. A wide variety ofsolid supports are known in the art, including those in the form ofresins, pins, or silicone chips. Preferably, the support is in the formof a resin. Examples of preferred resins include derivatized polystyreneresins, such as, WANG™ resin, MERRIFIELD™ resin, 4-methylbenzhydrylamine (i.e., MBHA) resin, RINK™ amide resin, RINK™ Amide MBHAresin, SIEBER™ resin, NOVASYN® TGR resin, and NOVASYN® TGA resin.

As discussed above, the hydroxyl group of the serine-like amino acid,which is to be converted to a halo group, may be in a suitably protectedform (e.g., as --OTBDMS), or in a free form (i.e., as --OH). Inembodiments where the reactant polypeptide comprises serine, homoserine,threonine, or other serine-like amino acids which are not to beconverted to halo groups (i.e., not the subject of halo-conversion), thehydroxyl groups of these amino acids are suitably protected prior tohalo-conversion, for example, with a tBu group (i.e., --C(CH₃)₃ ; as--OC(CH₃)₃).

Preferably, the thiol group (i.e., --SH) of any cysteine-like aminoacids of the reactant polypeptide are suitably protected prior tohalo-conversion. In preferred embodiments, such thiol groups areprotected with a Tr group (i.e., --C(C₆ H₅)₃ ; as --STr), or morepreferably, with a tBuS group (i.e., --SC(CH₃)₃ ; as --SSC(CH₃)₃).

Preferably, halo-conversion is performed using a reactant polypeptidewherein the side-chain functional groups are suitably protected. Forexample, in embodiments where the polypeptide comprises arginine, theguanidino group of arginine is protected, for example, with a Pmc, Mts,or Mtr group. In embodiments where the polypeptide comprises asparagineand glutamine, the carboxamide groups of asparagine and glutamine areprotected, for example, with a trityl (i.e., Tr) group. In embodimentswhere the polypeptide comprises aspartic and glutamic acid, the sidechain carboxyl groups of aspartic and glutamic acid are protected, forexample, with a tert-butyl (i.e., t-Bu) or cyclohexyl (i.e., cHx) group.In embodiments where the polypeptide comprises histidine, the imidazolegroup of histidine is protected, for example, with a trityl group. Inembodiments where the polypeptide comprises lysine, the epsilon-aminogroup of lysine is protected, for example, with a Boc, CBZ or 2-Cl--CBZgroup. In embodiments where the polypeptide comprises tryptophan, theindole nitrogen of tryptophan is protected, for example, with a tritylgroup. In embodiments where the polypeptide comprises tyrosine, thehydroxyl group of tyrosine is protected, for example, with a tritylgroup.

Halo-conversion may be performed using a reactant polypeptide where theterminal alpha-amino group is free (i.e., --NH₂ or --NR^(N) H) orsuitably protected. In preferred embodiments, the terminal alpha-aminogroup is protected, for example, with a Fmoc, Boc, or CBZ group (e.g.,as --NHFmoc, --NHBoc, --NHCBZ, respectively).

Preferably, halo-conversion is carried out using a molar excess of thephosphorus-based halogenation reagent. The molar excess may beconveniently calculated from the quantity of reactant polypeptide andthe quantity of phosphorus-based halogenation reagent. For embodimentswhere the reactant polypeptide is a supported polypeptide, the quantityof reactant polypeptide is determined from the substitution of the resin(i.e., how much polypeptide is theoretically attached to the resin). Inpreferred embodiments which employ triphenylphosphine dihalide as thephosphorus-based halogenation reagent, halo-conversion is morepreferably carried out using a three- to six-times molar excess oftriphenylphosphine dihalide, or a concentration of about 100 mg/mL oftriphenylphosphine dihalide reagent in a suitable solvent system.

Halo-conversion is carried out in a suitable solvent system, preferablyat about room temperature. Suitable solvents are those which do notcause any undesired side reactions. For those embodiments which employ aresin-supported reactant polypeptide, suitable solvents also preferablygive good solvation of the resin. Examples of suitable solvents includeACN (i.e., acetonitrile, CH₃ CN) and DCM (i.e., dichloromethane, CH₂Cl₂).

For super acid-labile resins, such as SIEBER™ resin, halo-conversion ispreferably carried out in the presence of a base, such as imidazole.

In those embodiments in which halo-conversion is performed using asupported polypeptide, it may be desirable to cleave the halogenatedpolypeptides from the solid support upon completion of haloconversion.The cleavage may be carried out using standard peptide synthesismethods. For example, the halogenated polypeptides may be detached froman MBHA resin using hydrogen fluoride with suitable scavangers, forexample, ethylene dithiol. Under these conditions, many protectinggroups, but not the Fmoc group (e.g., on the terminal alpha-aminogroup), may be removed from the polypeptides at the same time.Halo-polypeptides may be detached from a Wang resin usingtrifluoroacetic acid with suitable scavangers, for example, ethylenedithiol. Under these conditions, many protecting groups, but neither theFmoc group (e.g., on the terminal alpha-amino group) nor the tBuS group(e.g., on the thiol group of a cysteine-like amino acid), may be removedfrom the polypeptides at the same time.

D. Preparation of Cyclic Polypeptides

The present invention also pertains to methods for the preparation ofcyclic polypeptides, said cyclic polypeptides having at least onepolypeptide loop, said loop comprising a thioether linkage. Moreparticularly, such cyclic polypeptides may be prepared from halogenatedpolypeptides having (i) at least one haloalanine-like amino acid, saidhaloalanine-like amino acid having a halo group (i.e., --X where X isCl, Br, or I); and (ii) at least one cysteine-like amino acid, saidcysteine-like amino acid having a thiol group (i.e., --SH). Cyclicpolypeptides may be prepared from such halogenated polypeptides byintramolecular alkylation of the thiol group of a cysteine-like aminoacid by the halo group of a haloalanine-like amino acid under suitablebasic conditions to form a thioether linkage (i.e., "cyclization").

The term "cysteine-like amino acid" is used herein to refer toalpha-amino acids which may be represented by the formula HNR^(N)--CR^(S) R^(B) --COOH (as the free amino acid) or as --NH--CR^(S) R^(B)--C(═O)-- (when part of a polypeptide chain), wherein R^(N), R^(S) andR^(B) are substituents. R^(B) is --H or an organic substituent, forexample, an alkyl group having 1 to 6 carbon atoms, but more preferably--CH₃ or --H; and R^(N) is --H or an organic substituent, for example,an alkyl group having 1 to 6 carbon atoms, but more preferably --H. Twoor more of the substituents R^(N), R^(S) and R^(B) may together form asingle multivalent substituent. The substituent R^(S) (or a singlemultivalent substituent incorporating R^(S) and one or more of R^(N) andR^(B)) is a thiol-containing group. The term "thiol-containing group" isused herein to refer to organic moieties which comprise a thiol group(i.e., --SH). The alpha carbon of the cysteine-like amino acid may havechirality R or S.

In some preferred embodiments, R^(S) is a thiol-containing alkyl group.The term "thiol-containing alkyl group" is used herein to refer toorganic moieties which comprise a thiol group (i.e., --SH) and an alkylmoiety. The alkyl moiety preferably comprises from 1 to 10 carbon atoms,more preferably 1 to 5 carbon atoms, still more preferably 1 to 3 carbonatoms, most preferably 1 to 2 carbon atoms. The alkyl moiety may belinear, cyclic, or branched, but is preferably linear. Examples ofpreferred thiol-containing alkyl groups include those of the generalformula --(CH₂)_(z) SH where z is a natural number from 1 to 10, morepreferably 1 to 5, still more preferably 1 to 3, most preferably 1 to 2.Examples of more preferred thiol-containing alkyl groups include --CH₂SH (i.e., as in the case of cysteine) and --CH₂ CH₂ SH (i.e., as in thecase of homocysteine). Other examples of preferred thiol-containingalkyl groups include --CH(CH₃)SH and --C(CH₃)₂ SH (i.e., as in the caseof penicillamine). Still other examples of cysteine-like amino acidsinclude 4-mercaptoproline and 2-mercaptohistidine.

Different thioether linkages may be obtained by employing differenthalogenated polypeptides. For example, when the haloalanine-like aminoacid is obtained by halo-conversion of serine (R^(H) is --CH₂ --X), andthe cysteine-like amino acid is cysteine (R^(S) is --CH₂ --SH), thethioether linkage --CH₂ --S--CH₂ -- (i.e., MMTE;methylene-methylene-thioether) is obtained. Similarly, when thehaloalanine-like amino acid is obtained by halo-conversion of homoserine(R^(H) is --CH₂ CH₂ --X), and the cysteine-like amino acid ishomocysteine (R^(S) is --CH₂ CH₂ --SH), the thioether linkage --CH₂ CH₂--S--CH₂ CH₂ -- (i.e., EETE, ethylene-ethylene-thioether) is obtained.When the haloalanine-like amino acid is obtained by halo-conversion ofhomoserine (R^(H) is --CH₂ CH₂ --X), and the cysteine-like amino acid iscysteine (R^(S) is --CH₂ --SH), the thioether linkage --CH₂ CH₂ --S--CH₂-- (i.e., EMTE, ethylene-methylene-thioether) or --CH₂ --S--CH₂ CH₂ --(i.e., METE, methylene-ethylene-thioether) is obtained, according to therelative positions of the two amino acids. Similarly, when thehaloalanine-like amino acid is obtained by halo-conversion of serine(R^(H) is --CH₂ X), and the cysteine-like amino acid is homocysteine(R^(S) is --CH₂ CH₂ --SH), the thioether linkage --CH₂ --S--CH₂ CH₂ --(i.e., METE, methylene-ethylene-thioether) or --CH₂ CH₂ --S--CH₂ --(i.e., EMTE, ethylene-methylene-thioether) is obtained, according to therelative positions of the two amino acids.

Cyclization is effected by intramolecular alkylation of a thiol group bya halo group of a halogenated polypeptide having at least onehaloalanine-like amino acid and at least one cysteine-like amino acid,in a suitable basic medium. For example, cyclization can be achieved byreaction of the halogenated polypeptide with sodium carbonate (i.e., Na₂CO₃) in a suitable solvent.

Cyclization may be performed using a dissolved halogenated polypeptide(i.e., in solution) or using a supported halogenated polypeptide (e.g.,attached to a support material). For example, the halogenatedpolypeptide may be prepared, as describe above, by derivatizing areactant polypeptide (i.e., halo-conversion) while attached to a solidsupport. Cyclization may then be performed using the supportedhalogenated polypeptide, or alternatively, the halogenated polypeptidemay be cleaved from the support and cyclization performed using thedissolved halogenated polypeptide.

In those embodiments where cyclization is performed using a supportedhalogenated polypeptide wherein the thiol group of the cysteine-likeamino acid is in a protected form, it may be deprotected under suitableconditions. For example, a thiol group protected with a tBuS group maybe deprotected with tributyl phosphine (i.e., P(C₄ H₉)₃). A thiol groupprotected with a trityl group may be conveniently deprotected with 1%TFA (i.e., trifluoroacetic acid) in DCM (i.e., dichloromethane) plustrimethylsilane (i.e., SiH(CH₃)₃). Under these conditions, many othertypes of protecting groups remain intact. The cyclization reaction canbe effectively performed using a solvent mixture (1:1 v/v) ofacetonitrile (i.e., CH₃ CN) and water (i.e., H₂ O) with about 10-20mg/mL of sodium carbonate (i.e., Na₂ CO₃). Examples of preferredsupports for cyclization of a supported halogenated polypeptide includepoly(ethylene glycerol) resins, such as, NOVASYN® TGA and NOVASYN® TGRresins.

In those embodiments where the cyclization step is performed using adissolved halo-polypeptide (i.e., in solution), the thiol group of thecysteine-like amino acid may be deprotected (e.g., under the cleavageconditions). However, if necessary, it may be deprotected under suitableconditions. For example, a thiol group protected with a tBuS group maybe deprotected with tributyl phosphine (i.e., P(C₄ H₉)₃). To avoidintermolecular side reactions, high dilution of the halo-polypeptide insolution is necessary during cyclization. In solution, the cyclizationreaction can be effectively performed using a diluted polypeptidesolution (e.g., about 1 mg/mL) in a solvent mixture (1:1 v/v) ofacetonitrile (i.e., CH₃ CN) and water (i.e., H₂ O) with about 1 mg/mL ofsodium carbonate (i.e., Na₂ CO₃).

Thus, the cyclic polypeptides of the present invention may be preparedfrom reactant polypeptides having at least one serine-like amino acidand at least one cysteine-like amino acid by halo-conversion, first, andcyclization, second, as described above. More specifically, the cyclicpolypeptides of the present invention may be prepared from reactantpolypeptides having (i) at least one serine-like amino acid, saidserine-like amino acid having a hydroxyl group (i.e., --OH); and (ii) atleast one cysteine-like amino acid, said cysteine-like amino acid havinga thiol group (i.e., --SH) by (a) converting the hydroxyl group of saidserine-like amino acid to a halo group (i.e., --X where X is Cl, Br, orI) with the aid of a phosphorus-based halogenation reagent, thusyielding a haloalanine-like amino acid (i.e., "halo-conversion");followed by (b) intramolecular alkylation of the thiol group of acysteine-like amino acid by the halo group of a haloalanine-like aminoacid under suitable basic conditions to form a thioether linkage (i.e.,"cyclization"). The halo-conversion and cyclization steps are describedin detail above.

The halo-conversion step may be performed using a reactant polypeptidewhich is dissolved (i.e., in solution) or supported (e.g., attached to asupport material), as described above. Similarly, the cyclization stepmay be performed using a halogenated polypeptide which is dissolved(i.e., in solution) or supported (e.g., attached to a support material),as described above. In those embodiments in which halo-conversionemploys a supported polypeptide and in which the cyclization step is tobe performed in solution, the halogenated polypeptides may be cleavedfrom the solid support upon completion of the halo-conversion usingstandard peptide synthesis methods. Preferably, the halo-conversion stepis performed using a reactant polypeptide which is supported.

Many other modifications and variations of the invention as hereinbeforeset forth can be made without departing from the spirit and scopethereof and therefore only such limitations should be imposed as areindicated by the appended claims.

E. Examples

Several of the halogenated polypeptides and cyclic polypeptides of thepresent invention, and methods for preparing them, are described in thefollowing examples, which are offered by way of illustration and not byway of limitation.

For convenience, a number of chemical compounds are interchangeablyreferred to herein by their chemical name, chemical formula, and/or asuitable acronym. These include DCM (i.e., dichloromethane, CH₂ Cl₂);DMF (i.e., dimethylformamide, (CH₃)₂ NCHO); MeOH, (i.e., methanol, CH₃OH); EtOH (i.e., ethanol, CH₃ CH₂ OH); nPrOH (i.e., n-propanol, CH₃ CH₂CH₂ OH); TFA (i.e., trifluoroacetic acid, CF₃ COOH); DMS (i.e., dimethylsulfide, CH₃ SCH₃); ACN (i.e., acetonitrile, CH₃ CN); THF (i.e.,tetrahydrofuran, C₄ H₈ O); water (i.e., H₂ O); hydrogen fluoride (i.e.,HF); anisole (i.e., C₆ H₅ OCH₃); para-thiocresol (i.e., CH₃ --C₆ H₄--SH); diethyl ether (i.e., C₂ H₅ OC₂ H₅); sodium carbonate (i.e., Na₂CO₃); ethylene dithiol (i.e., HSCH₂ CH₂ SH); and tributylphosphine(i.e., P(C₄ H₉)₃).

The general analytical methods and characterization techniques used inthe present disclosure are identified below. ¹ H NMR spectra wererecorded on a Bruker AC300 spectrometer at 300 MHz. Chemical shifts wererecorded in parts per million (δ) relative to TMS (i.e.,tetramethylsilane, δ=0.0 ppm). Analytical HPLC analyses were performedon a Hewlett Packard liquid chromatography HP 1090 instrument fittedwith a Vydac C18 column (4.6×250 mm, 5 mm particle size). PreparativeHPLC was performed on Dynamax SD 200 system with a Vydac C18 column(22×250 mm, 10 mm particle size). The purity of peptide products wasanalyzed using two HPLC solvent systems: a trifluoroacetic acid (TFA)system or a triethylamine phosphate (TEAP) system. In the TFA system, agradient of 5-50% B over 20 min was used, where A was 0.1% (v/v) TFA/H₂O and B was 0.1% (v/v) TFA/ACN. In the TEAP system, a gradient of 5-60%B over 20 min was used, where A was 9:1 TEAP/ACN (v/v) and B was 4:6TEAP/ACN (v/v). TEAP buffer was prepared by adding 11 mL of concentratedphosphoric acid (i.e., H₃ PO₄, 85% w/v) to 900 mL of H₂ O and adjustingthe pH to 2.3 with triethylamine (i.e., N(C₂ H₅)₃, about 10 mL) and thenmade up to a volume of 1000 mL with more H₂ O.

All common amino acid derivatives were purchased from NovaBiochem orAdvanced ChemTech. N^(a)-(9-Fluorenylmethyoxycarbonyl)-O-t-butyldimethylsilyl-l-serine and N^(a)-(9-fluorenylmethyoxycarbonyl)-O-t-butyldimethylsilyl-d-serine wereobtained from Bachem Bioscience Inc. N^(a)-(9-Fluorenylmethyoxycarbonyl)-O-t-butyldimethylsilyl-l-homoserine wasprepared as described by Fisher (Tetrahedron Lett. (1992) 49:7605-7608).N^(a) -(9-Fluorenylmethyoxycarbonyl)-S-t-butylthio-l-homocysteine wasprepared according to the procedure of Wunsch et al. (Hoppe-Seyler's Z.Physiol. Chem. (1982), 363:1461-1464). Triphenylphosphine dichloride andtriphenylphosphine dibromide were purchased from Aldrich ChemicalCompany; their purities were monitored by ³¹ P NMR before use (Appel etal., Chem. Ber. (1976) 109:58-70). More preferably, triphenylphosphinedichloride was prepared fresh according to the procedure of Appel andScholer (Chem. Ber. (1977) 110:2382-2384).

The polypeptides used in the preparation of the cyclic polypeptides ofthe present invention were prepared using standard solid phase synthesismethods. The experimental details of two specific methods, denotesMethod A and Method B, which were used in the examples are describedbelow.

In Method A, the polypeptides were synthesized manually using standardFmoc solid phase chemistry (Stewart and Young, Solid Phase PeptideSynthesis, 2nd., Pierce Chemical Co,: Rockford, Ill., (1984); p 82;Fields and Noble, Int. J. Pept. Protein Res. (1990) 35:161-214). Duringeach cycle, the Fmoc group was removed by treatment with 20% piperidine(i.e., NHC₅ H₁₁) in DMF for 5 and 10 min. The peptide resin was thenwashed successively with DMF (twice), MeOH (twice), DMF (twice), andMeOH (twice). The amino acid was coupled to the resin using 3equivalents of the Fmoc-protected amino acid, 3 equivalents of DIC(i.e., N,N'-diisopropylcarbodiimide), and 3 equivalents of HOBt (i.e.,N-hydroxybenzotriazole) in DMF at 55° C. The coupling reaction wasmonitored by addition of indicator bromophenol blue (˜5 mL of a 0.05Msolution in DMF). Coupling continued until the disappearance of the bluecolor and formation of a yellow color. A typical single couplingrequired from 15 to 120 minutes, depending on the polypeptide sequenceand the amino acid residue to be coupled. The polypeptide resin waswashed successively with DMF (twice), MeOH (twice), DMF (twice), andMeOH (twice). The completion of the coupling was confirmed by aninhydrin test (Kaiser et al., Anal. Biochem. (1970) 34:595-598) anddouble coupling was performed if required.

In Method B, the polypeptides were synthesized using solid phasechemistry in an automated fashion on an Advanced ChemTech 357 MPSautomated synthesizer using Fmoc chemistry (Fields and Noble, Int. J.Pept. Protein Res. (1990) 35:161-214). A typical cycle for the couplingof an individual amino acid was as follows: (1) deprotection of theamino acid on the resin with 30% piperidine/DMF for 5 and 10 min; (2)washing successively with DMF, MeOH, DMF, and MeOH; (3) double couplingsof the amino acid, each with 6 equivalents of the Fmoc-protected aminoacid, 6 equivalents of DIC, and 6 equivalents of HOBt in DMF for 60 minat room temperature; (4) washing successively with DMF, MeOH, DMF, andMeOH. The resin was then transferred to the cleavage vessel and washedwith DCM and dried under vacuum.

EXAMPLE 1

Cyclization of (Fmoc)AGPHsLGVLGKLCPG (SEQ ID NO:53) to form 3G3-EMTE andN^(a) -Fmoc-3G3-EMTE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 2. The resin-bound fully protected peptide(Fmoc)AGP(TBDMS)HsLGVLG(CBZ)KL(tBuS)CPG(SEQ ID NO:53)-resin was preparedusing Method A on (Fmoc)-Gly-Wang resin (NovaBiochem, 0.50 g, 0.67mmol/g). Side chain functional groups were protected as follows: Cys(tBuS); Lys (CBZ); Hs (TBDMS). After completion of all couplings, thepeptide resin was washed with DCM (twice) and subsequently dried invacuo.

The TBDMS protected hydroxyl group (i.e., --OTBDMS) of homoserineresidue, Hs, was converted to chloro group (i.e., --Cl) by treatmentwith 6 equivalents of triphenylphosphine dichloride (i.e., (C₆ H₅)₃PCl₂) in DCM overnight at room temperature. The polypeptide resin waswashed successively with DMF (twice), MeOH (twice), DMF (twice), andMeOH (twice) and then dried in vacuo. The dried polypeptide resin wasthen treated with a 10:1:1:0.2 (v/v) mixture of HF, anisole, DMS, andpara-thiocresol for one hour at 0° C. After removal of HF in vacuo, theresidue was washed three times with diethyl ether to remove scavengersand extracted three times with 0.1% TFA in 1:1 (v/v) H₂ O/ACN. Thecombined filtrates were lyophilized and the crude polypeptide waspurified by preparative HPLC eluted at 10 mL/min with a linear gradientfrom 40 to 70% B over 40 minutes where A was 0.1% (v/v) TFA in H₂ O andB was 0.08% (v/v) TFA in ACN. The chloro-polypeptide was obtained as awhite powder after further lyophilization (153.5 mg, 28% yield;Analytical RP-HPLC: TFA system with a gradient of 20-80% B over 20 min:t_(R) 15.60 min; purity, 97.2%; MS (ESI): m/e (M+1) Calcd. for C₇₂ H₁₀₉N₁₅ O₁₇ SCl: 1523, obsd.: 1523).

The chloro-polypeptide (48.0 mg) was dissolved in 50 mL of a sodiumcarbonate (i.e., Na₂ CO₃, 1 mg/mL, pH ˜10.5) solution in ACN/water (1:1)at room temperature, under argon, for 36 hours with stirring. Thecyclization reaction was monitored by analytical HPLC. After thecompletion of cyclization, indicated by the disappearance of thestarting material, the solution was neutralized with TFA andlyophilized. The crude cyclic polypeptide material was purified usingpreparative HPLC eluted at 10 mL/min with a linear gradient from 10 to70% B over 40 minutes where A was 0.1% (v/v) TFA in H₂ O and B was 0.08%(v/v) TFA in ACN. Two cyclic polypeptides, N^(a) -Fmoc-3G3-EMTE and3G3-EMTE, were obtained (N^(a) -Fmoc-3G3-EMTE: 9.0 mg, 19% yield;Analytical RP-HPLC: TFA system with a gradient of 20-80% B over 20 min:t_(R) 15.58 min; purity, 97.0%; TEAP system: t_(R) 17.13 min; purity,94.0%; MS (ESI): m/e (M+Cs⁺) Calcd. for C₇₂ H₁₀₇ N₁₅ O₁₇ SCs: 1618.6744,obsd.: 1618.6763; 3G3-EMTE: 13.3 mg, 33% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.16 min; purity, 100%; TEAP system: t_(R) 12.85 min;purity, 100%; HRMS (ESI): m/e (M+Cs⁺) Calcd. for C₅₇ H₉₇ N₁₅ O₁₅ SCs:1396.6064, obsd.: 1396.6083).

EXAMPLE 2

Cyclization of AGPHsLGVLGKLCPG (SEQ ID NO:53) to form 3G3-EMTE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 3. The resin-bound fully protected peptide(CBZ)AGP(TBDMS)HsLGVLG(CBZ)KL(tBuS)CPG-resin was prepared by Method A on(Fmoc)-Gly-Wang resin (NovaBiochem, 0.50 g, 0.60 mmol/g). Side chainfunctional groups were protected as follows: Cys (tBuS); Lys (CBZ); Hs(TBDMS). The alpha-amino group of the polypeptide was protected with aCBZ group. After completion of all couplings, the peptide resin waswashed with DCM (twice) and dried in vacuo.

The TBDMS protected hydroxyl group (i.e., --OTBDMS) of homoserineresidue, Hs, was converted to chloro group (i.e., --Cl) by treatmentwith 6 equivalents of triphenylphosphine dichloride (i.e., (C₆ H₅)₃PCl₂) in DCM overnight at room temperature. The polypeptide resin waswashed successively with DMF (twice), MeOH (twice), DMF (twice), andMeOH (twice) and then dried in vacuo. The dried polypeptide resin wasthen treated with a 10:1:1:0.2 (v/v) mixture of HF, anisole, DMS, andpara-thiocresol for one hour at 0° C. After removal of HF in vacuo, theresidue was washed three times with diethyl ether to remove scavengersand extracted three times with 0.1% TFA in 1:1 (v/v) H₂ O/ACN. Thecombined filtrates were lyophilized and the crude polypeptide waspurified by preparative HPLC eluted at 10 mL/min with a linear gradientfrom 10 to 40% B over 40 minutes where A was 0.1% (v/v) TFA in H₂ O andB was 0.08% (v/v) TFA in ACN. The chloro-polypeptide was obtained as awhite powder after further lyophilization (100.5 mg, 22% yield;Analytical RP-HPLC: TFA system: t_(R) 16.48 min; purity, 95.1%; TEAPsystem: t_(R) 14.69 min; purity, 93.7%; MS (ESI): m/e (M+1) Calcd. forC₅₇ H₉₉ N₁₅ O₁₅ SCl: 1301, obsd.: 1301).

The chloro-polypeptide (18.5 mg) was dissolved in 20 mL of a sodiumcarbonate (i.e., Na₂ CO₃, 1 mg/mL, pH ˜10.5) solution in ACN/water (1:1)at room temperature, under argon, for 24 hours with stirring. Thecyclization reaction was monitored by analytical HPLC. After thecompletion of cyclization, indicated by the disappearance of thestarting material, the solution was neutralized with TFA andlyophilized. The crude cyclic polypeptide material was purified usingpreparative HPLC eluted at 10 mL/min with a linear gradient from 10 to40% B over 40 minutes where A was 0.1% (v/v) TFA in H₂ O and B was 0.08%(v/v) TFA in ACN. The cyclic polypeptides was obtained as a white powderafter further lyophilization (17.0 mg, 94% yield; Analytical RP-HPLC:

TFA system: t_(R) 15.16 min; purity, 100%; TEAP system: t_(R) 12.85 min;purity, 100%; HRMS (ESI): m/e (M+Cs⁺) Calcd. for C₅₇ H₉₇ N₁₅ O₁₅ SCs:1396.6064, obsd.: 1396.6083).

EXAMPLE 3

Cyclization of AGPSLGVLGKLCPG to form 3G3-MMTE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 4. The methods for polypeptide synthesis, chlorination, andcyclization described in Example 2, above, were adapted in this example.The same protecting group scheme for the side chain functional groupsand alpha-amino group was used in this example.

Using 0.50 g of (Fmoc)-Gly-Wang resin (NovaBiochem, 0.60 mmol/g) thechloro-polypeptide was obtained as a white powder after purification(105.5 mg, 23% yield; Analytical RP-HPLC: TFA system: t_(R) 15.99 min;purity, 92.5%; TEAP system: t_(R) 14.08 min; purity, 95.9%; MS (ESI):m/e (M+1) Calcd. for C₅₆ H₉₇ N₁₅ O₁₅ SCl: 1287, obsd.: 1287).

Using 50.0 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a mixture of two diastereomers (43.1 mg, 89% yield;Analytical RP-HPLC: TFA system: t_(R) 14.78 min; purity, 100%; TEAPsystem: t_(R) 11.98 min; purity, 100% with a shoulder; HRMS (ESI): m/e(M+Cs⁺) Calcd. for C₅₆ H₉₅ N₁₅ O₁₅ SCs: 1382.5907, obsd.: 1382.5919).

EXAMPLE 4

Cyclization of GPHsLGVLGKLHcPG (SEQ ID NO:54) to form 2G3-EETE

The methods for polypeptide synthesis, chlorination, and cyclizationdescribed in Example 2, above, were adapted in this example. The sameprotecting group scheme for the side chain functional groups andalpha-amino group was used in this example.

Using 1.0 g of (Fmoc)-Gly-Wang resin (Advanced ChemTech, 0.34 mmol/g)the chloro-polypeptide was obtained as a white powder after purification(72.0 mg, 17% yield; Analytical RP-HPLC: TFA system: t_(R) 16.89 min;purity, 100%; TEAP system: t_(R) 14.84 min; purity, 100%; HRMS (ESI):m/e (M+1) Calcd. for C₅₅ H₉₆ N₁₄ O₁₄ SCl: 1243.6640, obsd.: 1243.6692).

Using 25.0 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white powder (18.2 mg, 75% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.63 min; purity, 100%; TEAP system: t_(R) 13.81 min;purity, 100%; HRMS (ESI): m/e (M+1) Calcd. for C₅₅ H₉₅ N₁₄ O₁₄ S:1207.6873, obsd.: 1207.6827).

EXAMPLE 5

Cyclization of GPHsLGVLGKLCPG (SEQ ID NO:55) to form 2G3-EMTE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 5. The methods for polypeptide synthesis, chlorination, andcyclization described in Example 2, above, were adapted in this example.Side chain functional groups were protected as follows: Cys (tBuS); Lys(2-Cl--CBZ); Hs (TBDMS). The alpha-amino group of the peptide wasprotected with a CBZ group. The chlorination step was carried out usinga solution of triphenylphosphine dichloride (i.e., (C₆ H₅)₃ PCl₂) in DCM(130 mg/mL). The dried polypeptide resin was treated with a 10:1:1 (v/v)mixture of HF, DMS, and ethylene dithiol for one hour at 0° C. Thechloro-polypeptide was then purified using the methods of Example 2.

Using 0.5 g of (Fmoc)-Gly-Wang resin (Advanced ChemTech, 0.34 mmol/g)the chloro-polypeptide was obtained as a white powder after purification(82.6 mg, 39% yield; Analytical RP-HPLC: TFA system: t_(R) 16.12 min;purity, 88.4%; TEAP system: t_(R) 14.80 min; purity, 92.9%; MS (ESI):m/e (M+1) Calcd. for C₅₄ H₉₄ N₁₄ O₁₄ SCl: 1230, obsd.: 1230).

Using 36.5 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white powder (29.7 mg, 84% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.48 min; purity, 100%; TEAP system: t_(R) 13.58 min;purity, 100%; HRMS (ESI): m/e (M+1) Calcd. for C₅₄ H₉₃ N₁₄ O₁₄ S:1193.6717, obsd.: 1193.6674).

EXAMPLE 6

Cyclization of GPSLGVLGKLCPG (SEQ ID NO:32) to form 2G3-MMTE

The methods for polypeptide synthesis, chlorination, and cyclizationdescribed in Example 5, above, were adapted in this example. The sameprotecting group scheme for the side chain functional groups andalpha-amino group was used in this example.

Using 0.50 g of (Fmoc)-Gly-Wang resin (Advanced ChemTech, 0.34 mmol/g),the chloro-polypeptide was obtained as a white powder after purification(78.3 mg, 32% yield; Analytical RP-HPLC: TFA system: t_(R) 15.88 min;purity, 93.3%; TEAP system: t_(R) 14.30 min; purity, 100%; MS (ESI): m/e(M+1) Calcd. for C₅₃ H₉₂ N₁₄ O₁₄ SCl: 1216, obsd.: 1216).

Using 34.2 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a mixture of two diastereomers (28.9 mg, 87% yield;Analytical RP-HPLC:TFA system: t_(R) 14.90 min; purity, 100%; TEAPsystem: t_(R) 11.93 min, purity, 58.8% and 12.17 min, purity, 41.%; HRMS(ESI): m/e (M+1) Calcd. for C₅₃ H₉₁ N₁₄ O₁₄ S: 1179.6560, obsd.:1179.6610).

EXAMPLE 7

Cyclization of GPSLGVLGKLHcPG (SEQ ID NO:56) to form l-2G3-METE andd-2G3-METE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 6. The methods for polypeptide synthesis, chlorination, andcyclization described in Example 5, above, were adapted in this example.The same protecting group scheme for the side chain functional groupsand alpha-amino group was used in this example.

Using 1.0 g of (Fmoc)-Gly-Wang resin (Advanced ChemTech, 0.34 mmol/g)the chloro-polypeptide was obtained as a white powder after purification(25.2 mg, 6% yield; Analytical RP-HPLC: TFA system: t_(R) 16.29 min;purity, 100%; TEAP system: t_(R) 14.06 min; purity, 92.0%; HRMS (ESI):m/e (M+1) Calcd. for C₅₄ H₉₄ N₁₄ O₁₄ SCl: 1230, obsd.: 1230).

Using 34.2 mg of the chloro-polypeptide, two cyclic polypeptides wereobtained, the d-isomer and the l-isomer (d-isomer: 4.0 mg, 16% yield;Analytical RP-HPLC: TFA system: t_(R) 15.04 min; purity, 98.4%; TEAPsystem: t_(R) 12.66 min; purity, 91.6%; HRMS (ESI): m/e (M+Cs⁺) Calcd.for C₅₄ H₉₃ N₁₄ O₁₄ SCs: 1325.5693, obsd.: 1325.5703; and l-isomer: 7.0mg, 29% yield; Analytical RP-HPLC: TFA system: t_(R) 15.39 min; purity,85.3%; TEAP system: t_(R) 13.09 min; purity, 84.4%; HRMS (ESI): m/e(M+Cs⁺) Calcd. for C₅₄ H₉₃ N₁₄ O₁₄ SCs: 1325.5693, obsd.: 1325.5699).

EXAMPLE 8

Cyclization of GPCLGVLGKLHsPG (SEQ ID NO:57) to form 2G3-METE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 7. The methods for polypeptide synthesis, chlorination, andcyclization described in Example 5, above, were adapted in this example.The same protecting group scheme for the side chain functional groupswas used in this example. The alpha-amino group of the peptide wasprotected with a Boc group.

Using 1.0 g of (Fmoc)-Gly-Wang resin (Advanced ChemTech, 0.34 mmol/g)the chloropolypeptide was obtained as a white powder after purification(114.3 mg, 23% yield; Analytical RP-HPLC: TFA system: t_(R) 16.30 min;purity, 84.8%; TEAP system: t_(R) 14.59 min; purity, 85.6%; MS (ESI):m/e (M+1) Calcd. for C₅₄ H₉₄ N₁₄ O₁₄ SCl: 1230, obsd.: 1230).

Using 17.9 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white solid (6.3 mg, 36% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.39 min; purity, 85.3%; TEAP system: t_(R) 13.09 min;purity, 84.4%; HRMS (ESI): m/e (M+Cs⁺) Calcd. for C₅₄ H₉₃ N₁₄ O₁₄ SCs:1325.5693, obsd.: 1325.5699).

EXAMPLE 9

Cyclization of HsLGVLGKLC (SEQ ID NO:58) to form G3-EMTE

The methods for polypeptide synthesis, chlorination, and cyclizationdescribed in Example 8, above, were adapted in this example. The sameprotecting group scheme for the side chain functional groups and thealpha-amino group of the peptide was used in this example.

Using 0.45 g of MBHA resin (NovaBiochem, 0.42 mmol/g) thechloropolypeptide was obtained as a white powder after purification(158.2 mg, 73% yield; Analytical RP-HPLC: TFA system: t_(R) 15.83 min;purity, 100%; TEAP system: t_(R) 13.77 min; purity, 93.8%; MS (ESI): m/e(M+1) Calcd. for C₄₀ H₇₅ N₁₁ O₉ SCl: 920, obsd.: 920).

Using 50.0 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white solid (24.7 mg, 51% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.43 min; purity, 92.8%; TEAP system: t_(R) 12.94 min;purity, 94.4%; HRMS (ESI): m/e (M+1) Calcd. for C₄₀ H₇₄ N₁₁ O₉ S:885.5470, obsd.: 885.5491).

EXAMPLE 10

Cyclization of SLGVLGKLC (SEQ ID NO:38) to form G3-MMTE

The methods for polypeptide synthesis, chlorination, and cyclizationdescribed in Example 8, above, were adapted in this example. The sameprotecting group scheme for the side chain functional groups and thealpha-amino group of the peptide was used in this example.

Using 0.50 g of MBHA resin (NovaBiochem, 0.42 mmol/g) thechloropolypeptide was obtained as a white powder after purification(151.8 mg, 64% yield; Analytical RP-HPLC: TFA system: t_(R) 15.33 min;purity, 98.2%; TEAP system: t_(R) 13.40 min; purity, 98.4%; MS (ESI):m/e (M+1) Calcd. for C₃₉ H₇₃ N₁₁ O₉ SCl: 906, obsd.: 906).

Using 50.0 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white solid (23.9 mg, 49% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.13 min; purity, 97.1%; TEAP system: t_(R) 12.27 min;purity, 97.6%; HRMS (ESI): m/e (M+1) Calcd. for C₃₉ H₇₂ N₁₁ O₉ S:871.5313, obsd.: 871.5332).

EXAMPLE 11

Cyclization of HsLGVLGKLHc (SEQ ID NO:59) to form G3-EETE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 8. The resin-bound fully protected peptide(Boc)(TBDMS)HsLGVLG(Boc)KL(tBuS)Hc-resin was prepared by Method B onRink amide MBHA resin (NovaBiochem, 0.5 g, 0.5 mmol/g). Side chainfunctional groups were protected as follows: Hc (tBuS); Lys (Boc); Hs(TBDMS). The alpha-amino group was protected with a Boc group. Aftercompletion of all couplings, the peptide resin was transferred from thereaction vessel to the cleavage vessels. The resin was washed with DCM(twice) and dried in vacuo.

The chlorination of the polypeptide was carried out using a solution oftriphenylphosphine dichloride (i.e., P(C₆ H₅)₃ Cl₂) in DCM (200 mg/mL).The polypeptide resin was washed successively with DMF (twice), MeOH(twice), DMF (twice), and MeOH (twice) and then dried in vacuo. Thedried polypeptide resin was treated with 95% TFA aqueous solution forone hour at room temperature. After removal of TFA and water under astream of argon, the residue was washed three times with diethyl etherand then dissolved in 30 mL of 0.1% TFA in 1:1 (v/v) H₂ O/ACN. To removethe tBuS protecting group of homocysteine residue, 0.75 mLtributylphosphine (i.e., P(C₄ H₉)₃) was added to the crude polypeptidesolution and stirred overnight at room temperature. The reaction mixturewas lyophilized and the crude polypeptide was purified by preparativeHPLC eluted at 10 mL/min with a linear gradient from 10 to 40% B over 40minutes where A was 0.1% (v/v) TFA in H₂ O and B was 0.08% (v/v) TFA inACN. The chloro-polypeptide was obtained as a white powder afterlyophilization (162.5 mg, 70% yield; Analytical RP-HPLC: TFA system:t_(R) 16.36 min; purity, 100%; TEAP system: t_(R) 14.74 min; purity,88.1%; HRMS (ESI): m/e (M+1) Calcd. for C₄₁ H₇₇ N₁₁ O₉ SCl: 934.5315,obsd.: 934.5361).

The cyclization was carried out according to the method in Example 2.Using 53.0 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white solid (24.2 mg, 48% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.61 min; purity, 97.1%; TEAP system: t_(R) 13.23 min;purity, 98.0%; HRMS (ESI): m/e (M+1) Calcd. for C₄₁ H₇₆ N₁₁ O₉ S:899.5626, obsd.: 899.5646).

EXAMPLE 12

Cyclization of SLGVLGKLHc (SEQ ID NO:60) to form G3-METE

The methods for polypeptide synthesis, chlorination, and cyclizationdescribed in Example 11, above, were adapted in this example. The sameprotecting group scheme for the side chain functional groups and thealpha-amino group of the peptide was used in this example.

Using 0.50 g of Rink amide MBHA resin (NovaBiochem, 0.50 mmol/g) thechloropolypeptide was obtained as a white powder after purification(57.5 mg, 25% yield; Analytical RP-HPLC: TFA system: t_(R) 15.93 min;purity, 97.4%; TEAP system: t_(R) 13.81 min; purity, 95.4%; HSMS (ESI):m/e (M+1) Calcd. for C₄₀ H₇₅ N₁₁ O₉ SCl: 920.5158, obsd.: 920.5206).

Using 17.3 mg of the chloro-polypeptide, the cyclic polypeptide wasobtained as a white solid (8.5 mg, 52% yield; Analytical RP-HPLC: TFAsystem: t_(R) 15.65 min; purity, 94.8%; TEAP system: t_(R) 13.15 min;purity, 93.4%; HRMS (ESI): m/e (M+1) Calcd. for C₄₀ H₇₄ N₁₁ O₉ S:885.5470, obsd.: 885.5488).

EXAMPLE 13

Cyclization of GPSLILAPDRC (SEQ ID NO:48) to form CB10-MMTE

The resin-bound fully protected peptide(Boc)GP(Tr)SLILAP(tBu)D(Pmc)R(tBuS)C-resin was synthesized using MethodA on MBHA resin (NovaBiochem, 2.0 g, 0.6 mmol/g). Before the firstcoupling, the MBHA resin was neutralized with 20% piperidine (˜5 mL/g)in DMF for 5 min and then washed successively with DMF (twice), MeOH(twice), DMF (twice), and MeOH (twice). Side chain functional groupswere protected as follows: Arg (Pmc); Asp (tBu); Cys (tBuS); Lys (CBZ);and Ser (Tr). After completion of the polypeptide synthesis, the tritylprotecting group of the serine residue was selectively removed bytreatment five times with 1% TFA in DCM/MeOH (1:1 v/v) each for 30minutes. The peptide resin was washed with DCM (twice) and subsequentlydried in vacuo to yield 3.38 g of the resin-bound polypeptide.

The free hydroxyl group (i.e., --OH) of the serine residue, S, wasconverted to bromo group (i.e., --Br) by treatment of the resin-boundpolypeptide (0.5 g, 0.044 mmol) with triphenylphosphine dibromide (i.e.,(C₆ H₅)₃ PBr₂, 172 mg, 0.407 mmol) and DIEA (i.e., ((CH₃)₂ CH)₂ NCH₂CH₃, diisopropylethylamine, 25 μl, 0.138 mmol) in 4 mL ACN overnight atroom temperature. The polypeptide resin was washed successively with DMF(twice), MeOH (twice), DMF (twice), and MeOH (twice) and subsequentlydried in vacuo. The dried polypeptide resin was then cleaved/deprotectedwith a 10:1:1:0.2 (v/v) mixture of HF, anisole, DMS, and para-thiocresolfor one hour at 0° C. After removal of HF in vacuo, the residue waswashed three times with diethyl ether to remove scavengers and extractedthree times with 0.1% TFA in 1:1 (v/v) H₂ O/ACN. The combined filtrateswere lyophilized and the crude polypeptide was purified by preparativeHPLC eluted at 10 mL/min with a linear gradient from 10 to 40% B over 40minutes where A was 0.1% (v/v) TFA in H₂ O and B was 0.08% (v/v) TFA inACN. The bromo-polypeptide was obtained as a white powder after furtherlyophilization (12.7 mg, 24% yield; MS (ESI): m/e (M+1) Calcd. for C₄₉H₈₅ N₁₅ O₁₃ SBr: 1203, 1205, obsd. 1203, 1205).

The bromo-polypeptide (12.7 mg) was dissolved in 70 mL of an aqueoussolution of sodium carbonate (i.e., Na₂ CO₃) of pH ˜10.5 for two daysunder argon. The cyclization reaction was monitored by analytical HPLC.After the completion of cyclization, indicated by the disappearance ofthe starting material, the solution was neutralized with TFA andlyophilized. The crude peptide was purified by preparative HPLC elutedat 10 mL/min with a linear gradient from 10 to 40% B over 40 minuteswhere A was 0.1% (v/v) TFA in H₂ O and B was 0.08% (v/v) TFA in ACN. Thecyclic polypeptide was obtained as a white powder after furtherlyophilization (3.2 mg, 27% yield; MS (ESI): m/e (M+1) Calcd. for C₄₉H₈₄ N₁₅ O₁₃ S: 1123, obsd. 1123).

EXAMPLE 14

Cyclization of HsL(N^(a) MeGly)(d-V)(d-L)AKLC (SEQ ID NO:62) to formAG3-EMTE

A reaction scheme illustrating the synthesis in this example is shown inFIG. 9. The resin-bound fully protected peptide (Boc)(TBDMS)HsL(N^(a)MeGly)(d-V)(d-L)A(Boc) KL(tBuS)C-resin was prepared by Method B onNOVASYN® TGR resin (NovaBiochem, 1.0 g, 0.2 mmol/g). The notations d-Vand d-L refer to d-valine and d-leucine, respectively. Side chainfunctional groups were protected as follows: Hs(TBDMS); Lys (Boc); Cys(tBuS). The alpha-amino group was protected with a Boc group. Aftercompletion of all couplings, the peptide resin was transferred from thereaction vessel to the cleavage vessels, and the resin washed with DCM(twice) and dried in vacuo.

The chlorination of the supported polypeptide was carried out using 6equivalents of triphenylphosphine dichloride (i.e., P(C₆ H₅)₃ Cl₂) inDCM. The chlorination was completed after two hours as determined bycleaving a small portion of the peptide resin with 95% TFA aqueoussolution for one hour at room temperature and analyzing the cleavedpeptide by HPLC. The polypeptide resin was washed successively with DMF(twice), MeOH (twice), DMF (twice), MeOH (twice), and DCM (twice).

The tBuS protecting group on the cysteine residue was removed bytreatment of the supported chlorinated polypeptide with 299 μl oftributylphosphine (i.e., P(C₄ H₉)₃) in 10 mL of nPrOH/DMF/H₂ O (5:3:2)for one hour at room temperature. Afterward, the resin was washedsuccessively with DMF (twice), MeOH (twice), DMF (twice), and MeOH(twice).

The on-resin cyclization was carried out in 10 mL of sodium carbonatesolution (i.e., Na₂ CO₃, 20 mg/mL) in ACN/H₂ O (1:1) for 48 hours atroom temperature. After the completion of the cyclization, indicated bythe absence of yellow color in the Ellman test (see, Ellman, Arch.Biochem, Biophys. (1959) 82:70), the resin was washed successively withDMF (twice), MeOH (twice), and DCM (twice), and subsequently dried invacuo. The supported cyclic polypeptide was cleaved from the driedpolypeptide resin by treatment with 95% TFA aqueous solution for onehour at room temperature. After removal of TFA and water in vacuo, thecrude cyclic polypeptide was purified using preparative HPLC eluted at10 mL/min with a linear gradient from 10 to 40% B in A over 40 minuteswhere A was 0.1% (v/v) TFA in H₂ O and B was 0.08% (v/v) TFA in ACN. Thecyclic polypeptide was obtained as a white powder after lyophilization(5.7 mg, 3% yield; Analytical RP-HPLC: TFA system: t_(R) 14.54 min;purity 84.9%; TEAP system: t_(R) 10.50 min; purity 86.9%; HRMS (ESI):m/e (M+Cs⁺) Calcd. for C₄₂ H₇₈ N₁₁ O₉ SCs: 1044.4681, obsd. 1044.4653).

Examples 15 through 18 demonstrate the haloconversion of the serine-likeamino acid, homoserine, when present in a polypeptide containing variousother naturally occurring amino acids.

EXAMPLE 15

Chlorination of HsLRSLGEMC (SEQ ID NO:62)

The method for polypeptide synthesis in Example 14, above, was adaptedin this example. Side chain functional groups were protected as follows:Hs (TBDMS); Arg (Pmc); Ser (tBu); Cys (tBuS). The alpha-amino group ofthe peptide was protected with a Boc group.

The chlorination of the polypeptide was carried out using 3 equivalentsof freshly prepared triphenylphosphine dichloride (i.e., P(C₆ H₅)₃ Cl₂)in DCM for one hour. The polypeptide resin was washed successively withDMF (twice), MeOH (twice), and DCM (twice) and subsequently dried invacuo. The chloropolypeptide was cleaved from the resin by treatmentwith 95% TFA aqueous solution at room temperature for one hour. Afterremoval of the solvents in vacuo, the purity of the crude product wasanalyzed by RF-HPLC on a C-18 column eluted at 1 mL/min with a lineargradient from 20 to 80% B in A over 20 minutes where A was 0.1% (v/v)TFA in H₂ O and B was 0.08% (v/v) TFA in ACN. The crude chloropeptidehas two major components: the starting material (t_(R) 8.84 min, 25.2%;MS (ESI): m/e (M+1) Calcd. for C₄₄ H₈₂ N₁₃ O₁₃ S₃ : 1906, obsd.: 1096)and the chloropeptide (t_(R) 9.26 min, 34.8%; MS (ESI): m/e (M+1) Calcd.for C₄₄ H₈₁ N₁₃ O₁₂ S₃ Cl: 1114, obsd.: 1114).

EXAMPLE 16

Chlorination of HsLWFLGDLC (SEQ ID NO:63)

The methods for polypeptide synthesis and chlorination in Example 15,above, were adapted in this example. Side chain functional groups wereprotected as follows: Hs (TBDMS); Trp (Boc); Asp (tBu); Cys (tBuS). Thealpha-amino group of the peptide was protected with a Boc group.

After the chlorination and the cleavage, the crude chloropeptide wasanalyzed by RF-HPLC and only one major peak was observed (t_(R) 14.40min, 80.6%, MS (ESI): m/e (M+1) Calcd. for C₅₅ H₈₃ N₁₁ O₁₁ S₂ Cl: 1172,obsd.: 1172).

EXAMPLE 17

Chlorination of HsHNLGQLC (SEQ ID NO:64)

The methods for polypeptide synthesis and chlorination in Example 15,above, were adapted in this example. Side chain functional groups wereprotected as follows: Hs (TBDMS); His (Tr); Asn (Tr), Gln (Tr); Cys(tBuS). The alpha-amino group of the peptide was protected with a Bocgroup.

After the chlorination and the cleavage, the purity of the crude productwas determined by analytical RF-HPLC and two major components wereobserved: the starting material (t_(R) 8.91 min, 32.5%; MS (ESI): m/e(M+1) Calcd. for C₄₆ H₈₁ N₁₄ O₁₂ S₂ : 1085, obsd.: 1085) and thechloropeptide (t_(R) 9.33 min, 57.2%; MS (ESI): m/e (M+1) Calcd. for C₄₆H₈₀ N₁₄ O₁₁ S₂ Cl: 1103, obsd.: 1103).

EXAMPLE 18

Chlorination of HsYGTLGKLC (SEQ ID NO:65)

The methods for polypeptide synthesis and chlorination in Example 15,above, were adapted in this example. Side chain functional groups wereprotected as follows: Hs (TBDMS); Tyr (tBu); Thr (tBu); Lys (Boc); Cys(tBuS). The alpha-amino group of the peptide was protected with a Bocgroup.

After the chlorination and the cleavage, the purity of the crude productwas determined by analytical RF-HPLC and one major components wasobserved: the chloropeptide (t_(R) 9.35 min, 71.6%; MS (ESI): m/e (M+1)Calcd. for C₄₆ H₇₉ N₁₁ O₁₁ S₂ Cl: 1060, obsd.: 1060).

EXAMPLE 19

Determination of Binding Affinity of Thioether Cyclic Polypeptide toAnticardiolipin Antibody

The binding affinities of a number of the thioether cyclic polypeptidesof the present invention to anticardiolipin antibody were determined bya competitive ELISA (i.e., enzyme-linked immunosorbent assay) andcompared with binding affinities of the corresponding disulfide cyclicpolypeptides (e.g., 3G3, 2G3, and G3).

Of 96 wells of a flat-bottom Immulon I microtiter plate (Dynatech Labs,Alexandria, Va.), 94 wells were coated with 50 mg cardiolipin per wellin 30 mL of ethanol. The remaining two wells were used as controls andeach received 30 mL of ethanol. After overnight evaporation at 4° C.,the plate was blocked for 2 hours at room temperature with 200 mL of 5%(w/v) fish gelatin in phosphate buffered saline (i.e., PBS, 0.15M NaCland 0.01M Na₂ HPO₄ at pH 7.2). The plate was washed five times in Trisbuffered saline (i.e., TBS, 0.15M NaCl and 0.05M Tris-HCl at pH 8.5).Then, β₂ -glycoprotein I (i.e., β₂ -GPI) was added as 100 mL/well of2.3% (v/v) IgG-depleted human serum (Sigma Chemical Co.) and incubatedfor 2 hours at room temperature.

During this incubation, peptide solutions (around 2 mg/mL) were preparedby dissolving thioether cyclic peptides in 3% fish gelatin in TBS. Theserums of patient ACA-6501, who has a GPL (i.e., IgG Phospholipid) scoreof 1500, and patient ACA-6701, who has a GPL score of 102, were dilutedabout 40-fold in 3% fish gelatin in TBS-PBS (1:1). Variable amounts ofeach of peptides were combined with 22 mL of each of the diluted humanserums and then made up to the final volume of 220 mL with 3% fishgelatin in TBS-PBS (1:1). For each peptide, at least four peptideconcentrations were employed and each data point was determined induplicate.

After 5 washes with TBS, 100 mL of the peptide/human serum solution wasadded and the microplate was agitated at 40 rpm in an orbital shaker(American Scientific, Rotator V) for one hour at room temperature. Theplate was washed extensively with TBS (5 times) and 100 mL of diluted(1/1000) alkaline phosphatase-conjugated goat anti-human IgG (Zymed,South San Francisco, Calif.) in 0.5% (w/v) BSA-TBS was added to eachwell (i.e., bovine serum albumin, BSA). The plate was then incubated forone hour at room temperature followed by addition of 100 mL/well of PPMPsolution (3 g/L phenolphthalein monophosphate plus 26.7 g/L2-amino-2-methyl-1-propanol in water). The plate was allowed to developat room temperature for 21 min and the reaction was stopped by adding 50mL of 0.2M Na₂ HPO₄ (Mallinckrodt) to each well. Blanks consisted ofprotein-coated wells that received similar treatment except human serumwas not added to these wells. The plate was read at 550 nm using amicroplate reader (Bio-Tek Instruments, Model EL 311).

Absorbance vs. amount of peptide added was plotted using Graph Pad Prism(Graph Pad Software, Inc.). The amount of peptide that inhibited thehuman serum's binding by 50%, known as IC₅₀, was calculated from thegraph at the intersection of half-maximal absorbance with amount ofpeptide added.

The results are shown in Table 1. In general, the thioether analogs havesimilar biological activities in comparison with the correspondingdisulfide cyclic peptides. Interestingly, one of the thioester cyclicpeptides in the series of G3 peptides, G3-EMTE, is more active than thedisulfide peptide G3. In the case of the patient ACA-6501, G3-EMTE isabout twice as active as G3.

                  TABLE 1                                                         ______________________________________                                                        IC.sub.50  (μM)                                            Cyclic Polypeptide                                                                            ACA-6501  ACA-6701                                            ______________________________________                                        3G3             857       491                                                 3G3-EMTE        ˜1119                                                                             not det'd.                                          3G3-MMTE        ˜1051                                                                             ˜1051                                         2G3             190       165                                                 2G3-EETE        ˜704                                                                              480                                                 2G3-EMTE        461       377                                                 d-2G3-METE      100       436                                                 l-2G3-METE      209       486                                                 2G3-MMTE        >>678     >>678                                               G3              111       44                                                  G3-EETE         89        40                                                  G3-EMTE         52        34                                                  G3-METE         104       36                                                  G3-MMTE         126       57                                                  ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 65                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 2..4                                                            (D) OTHER INFORMATION: /note= "thioether linkage to form                      cyclic polypeptide"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       XaaXaaXaaXaaXaa                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 4..12                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaGlyProCysLeuGlyValLeuGlyLysLeuCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 4..12                                                           (D) OTHER INFORMATION: /note= "methyl-methyl thioether                        bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaGlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 4..12                                                           (D) OTHER INFORMATION: /note= "ethyl-methyl thioether                         bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaGlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 4..12                                                           (D) OTHER INFORMATION: /note= "methyl-ethyl thioether                         bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaGlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 4..12                                                           (D) OTHER INFORMATION: /note= "ethyl-ethyl thioether                          bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaGlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 3..11                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GlyProCysLeuGlyValLeuGlyLysLeuCysProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "methyl-methyl thioeither                       bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "ethyl-methyl thioeither                        bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "methyl-ethyl thioeither                        bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "ethyl-ethyl thioeither                         bridge"                                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 1..9                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CysLeuGlyValLeuGlyLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "methyl-methyl thioether                        bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxy protecting group (amide)"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ethyl-methyl thioether                         bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "methyl-ethyl thioether                         bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ethyl-ethyl thioether                          bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 1..9                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CysLeuGlyValLeuAlaLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "methyl-methyl thioether                        bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      XaaLeuGlyValLeuAlaLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ethyl-methyl thioether                         bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      XaaLeuGlyValLeuAlaLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "methyl-ethyl thioether                         bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      XaaLeuGlyValLeuAlaLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION: /note= "ethyl-ethyl thioether                          bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      XaaLeuGlyValLeuAlaLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 3..11                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GlyProCysLeuIleLeuAlaProAspArgCys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "methyl-methyl thioether                        bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GlyProXaaLeuIleLeuAlaProAspArgXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "ethyl-methyl thioether                         bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GlyProXaaLeuIleLeuAlaProAspArgXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "methyl-ethyl thioether                         bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GlyProXaaLeuIleLeuAlaProAspArgXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 3..11                                                           (D) OTHER INFORMATION: /note= "ethyl-ethyl thioether                          bridge"                                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyProXaaLeuIleLeuAlaProAspArgXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AlaGlyProSerLeuGlyValLeuGlyLysLeuCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2Xel=                                                                       /note= "methyl halide"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AlaGlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      AlaGlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2Xel=                                                                       /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AlaGlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 12                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      AlaGlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GlyProSerLeuGlyValLeuGlyLysLeuCysProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2Xel=                                                                       /note= "methyl halide"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2Xel=                                                                       /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GlyProCysLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      SerLeuGlyValLeuGlyLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2Xel=                                                                       /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      XaaLeuGlyValLeuGlyLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      XaaLeuGlyValLeuGlyLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2Xel=                                                                       /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc-NH2                                                                /note= "homocysteine with carboxyl protecting group (amid                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       CH2CH2X                                                                       /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc-NH2                                                                /note= "homocysteine with carboxyl protecting group                           (amide)"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      SerLeuGlyValLeuAlaLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               CH2XOTHER INFORMATION: /label=                                                /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="MeGly"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Val"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Leu"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      XaaLeuXaaXaaXaaAlaLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               CH2CH2XER INFORMATION: /label=                                                /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="MeGly"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Val"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Leu"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group (amide)"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      XaaLeuXaaXaaXaaAlaLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               CH2XOTHER INFORMATION: /label=                                                /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="MeGly"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Val"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Leu"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc-NH2                                                                /note= "homocysteine with carboxyl protecting group                           (amide)"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      XaaLeuXaaXaaXaaAlaLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               CH2CH2XER INFORMATION: /label=                                                /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="MeGly"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Val"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Leu"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc-NH2                                                                /note= "homocysteine with carboxyl protecting group                           (amide)"                                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      XaaLeuXaaXaaXaaAlaLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GlyProSerLeuIleLeuAlaProAspArgCys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               CH2XOTHER INFORMATION: /label=                                                /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      GlyProXaaLeuIleLeuAlaProAspArgCys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               CH2CH2XER INFORMATION: /label=                                                /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              NH2 OTHER INFORMATION: /label=                                                /note= "carboxyl protecting group"                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GlyProXaaLeuIleLeuAlaProAspArgCys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               CH2XOTHER INFORMATION: /label=                                                /note= "methyl halide"                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc-NH2                                                                /note= "homocysteine with carboxyl protecting group"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GlyProXaaLeuIleLeuAlaProAspArgXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               CH2CH2XER INFORMATION: /label=                                                /note= "ethyl halide"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc-NH2                                                                /note= "homocysteine with carboxyl protecting group"                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GlyProXaaLeuIleLeuAlaProAspArgXaa                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      AlaGlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GlyProXaaLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GlyProXaaLeuGlyValLeuGlyLysLeuCysProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GlyProSerLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 11                                                              (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GlyProCysLeuGlyValLeuGlyLysLeuXaaProGly                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      XaaLeuGlyValLeuGlyLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      XaaLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hc                                                                    /note= "homocysteine"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      SerLeuGlyValLeuGlyLysLeuXaa                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="MeGly"                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Val"                                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "D-Leu"                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      XaaLeuXaaXaaXaaAlaLysLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      XaaLeuArgSerLeuGlyGluMetCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      XaaLeuTrpPheLeuGlyAspLeuCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      XaaHisAsnLeuGlyGlnLeuCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /label= Hs                                                                    /note= "homoserine"                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      XaaTyrGlyThrLeuGlyLysLeuCys                                                   15                                                                            __________________________________________________________________________

I claim:
 1. A method for the preparation of a cyclic polypeptide, saidcyclic polypeptide having a polypeptide loop, said loop comprising athioether linkage;from a reactant polypeptide, said reactant polypeptidehaving a cysteine-like amino acid, said cysteine-like amino acid havinga thiol group, and a serine-like amino acid, said serine-like amino acidhaving an hydroxyl group; said method comprising the steps of:(a)converting said hydroxyl group of said serine-like amino acid to a halogroup with the aid of a phosphorus-based halogenation reagent to yield ahaloalanine-like amino acid, and thus form a halogenated polypeptide;and (b) intramolecularly reacting said halo group of saidhaloalanine-like amino acid of said halogenated polypeptide with saidthiol group of said cysteine-like amino acid of said halogenatedpolypeptide under basic conditions to form said thioether linkage. 2.The method of claim 1, wherein said phosphorus-based halogenationreagent comprises a reagent selected from the group consisting oftriphenylphosphine dihalide, triphenylphosphite dihalide, mixtures oftriphenylphosphine and a halohydrocarbon compound, and mixtures oftriphenylphosphite and a halohydrocarbon compound.
 3. The method ofclaim 1, wherein said basic conditions are provided by the addition ofsodium carbonate.
 4. The method of claim 1, wherein said reactantpolypeptide is provided in a dissolved form.
 5. The method of claim 1,wherein said reactant polypeptide is provided in a supported form; saidconversion step (a) is performed using said supported reactantpolypeptide; said halogenated polypeptide produced in step (a) iscleaved from its support to yield a dissolved halogenated polypeptide,prior to carrying out step (b); and said reaction step (b) is performedusing said dissolved halogenated polypeptide.
 6. The method of claim 1,wherein said reactant polypeptide is provided in a supported form; saidconversion step (a) is performed using said supported reactantpolypeptide to yield a supported halogenated polypeptide; and saidreaction step (b) is performed using said supported halogenatedpolypeptide.
 7. The method of claim 1, wherein said phosphorus-basedhalogenation reagent comprises triphenylphosphine dichloride.
 8. Themethod of claim 1, wherein said phosphorus-based halogenation reagentcomprises triphenylphosphine dibromide.
 9. The method of claim 1,wherein said phosphorus-based halogenation reagent comprises a mixtureof triphenylphosphine and carbon tetrachloride.
 10. The method of claim1, wherein a molar excess of said phosphorus-based halogenation reagent,in relation to said reactant polypeptide, is employed.
 11. The method ofclaim 1, wherein said hydroxyl group of said serine-like amino acid isin a protected form.
 12. The method of claim 1, wherein said hydroxylgroup of said serine-like amino acid is in a protected form as atert-butyldimethylsilyl ether group.
 13. The method of claim 1, whereinsaid reactant polypeptide is in a supported form.
 14. A method for thepreparation of a halogenated polypeptide, said halogenated polypeptidehaving a haloalanine-like amino acid, said haloalanine-like amino acidhaving a halo group --X wherein X is Cl, Br, or I;from a reactantpolypeptide, said reactant polypeptide having a serine-like amino acid,said serine-like amino acid having an hydroxyl group; said methodcomprising the step:(a) converting said hydroxyl group of saidserine-like amino acid to a halo group with the aid of aphosphorus-based halogenation reagent to yield a haloalanine-like aminoacid.
 15. The method of claim 14, wherein said phosphorus-basedhalogenation reagent comprises a reagent selected from the groupconsisting of triphenylphosphine dihalide, triphenylphosphite dihalide,mixtures of triphenylphosphine and a halohydrocarbon compound, andmixtures of triphenylphosphite and a halohydrocarbon compound.
 16. Themethod of claim 14, wherein said phosphorus-based halogenation reagentcomprises triphenylphosphine dichloride.
 17. The method of claim 14,wherein said phosphorus-based halogenation reagent comprisestriphenylphosphine dibromide.
 18. The method of claim 14, wherein saidphosphorus-based halogenation reagent comprises a mixture oftriphenylphosphine and carbon tetrachloride.
 19. The method of claim 14,wherein a molar excess of said phosphorus-based halogenation reagent, inrelation to said reactant polypeptide, is employed.
 20. The method ofclaim 14, wherein said hydroxyl group of said serine-like amino acid isin a protected form.
 21. The method of claim 14, wherein said hydroxylgroup of said serine-like amino acid is in a protected form as atert-butyldimethylsilyl ether group.
 22. The method of claim 14, whereinsaid reactant polypeptide is in a dissolved form.
 23. The method ofclaim 14, wherein said reactant polypeptide is in a supported form.